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Nuclear magnetic resonance studies on the interaction of metal ions with adenine nucleotides and substrates binding to adenylate kinase

Shyy, Yeun-Jund, Ph.D.

The Ohio State University, 1987

UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 PLEASE NOTE:

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University Microfilms International NUCLEAR MAGNETIC RESONANCE STUDIES ON THE INTERACTION OF METAL

IONS WITH ADENINE NUCLEOTIDES AND SUBSTRATES BINDING

TO ADENYLATE KINASE

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of the Ohio State University

By

Yeun-Jund Shyy, B. S.

*****

The Ohio State University

1987

Dissertation Committee: Approved by

M.-D. Tsai

M. H. Klapper 'TX-K: Adviser R. M. Mayer Department of Chemistry To My Parents

i i ACKNOWLEDGEMENTS

I wish to express special appreciation to Dr. Ming-Daw Tsai, my advisor and friend. His instruction throughout the research and the assistance given to me has made this dissertation possible. I also wish to thank the other members of my advisory committee, Drs.

Michael H. Klapper and Robert M. Mayer, for their helpful comments.

A sincere thanks goes to the colleagues in Tsai's group for providing the support and encouragement during the past few years. Finally, I am particularly grateful to my parents and my brother; this final stage would not have been possible were it not for the support they have given.

m VITA

August 31, 1956 Born, Hsin-Chu, Taiwan, Republic of China

1979 B.S., Fu-Jen Catholic Univ. Taipei, Taiwan, R.O.C.

1981-1987 Teaching Associate and Research Associate, Department of Chemistry, The Ohio State Univ. Columbus, Ohio

PUBLICATIONS

Phospholipids Chiral at Phosphorus. Absolute Configuration of Chiral

Thiophoslipids and Stereospecificty of Phospholipase D. (1984)

Biochemistry, 23, 1661-1667.

Mechanism of Adenylate Kinase. 1. Use of ^ 0 NMR to Study the Binding

Properties of Substrates (1985) Am. Chem. Soc. 107, 2814-2815.

Metal-Nucleotide Interactions. 3. ^ 0 , 3 1 P, *H NMR Studies on the

Interaction of Sc(III), La(III), and Lu(III) with Adenosine 5'-

Triphosphate (1985) J. Am. Chem. Soc. 107, 3478-3484.

FIELDS OF STUDIES

Major Field: Biochemistry, Bioorganic Chemistry.

iv TABLE OF CONTENTS

DEDICATION...... ii

ACKNOWLEDGMENTS...... 111

VITA ...... iv

LIST OF TABLES ...... v ii

LIST OF FIGURES ...... v iii

ABBREVIATIONS AND SYMBOLS ...... x11

CHAPTER PAGE

I . INTRODUCTION ...... 1

The Interaction of M(II) with ADP and ATP .... 1 The Interaction of M(III) with ATP ...... 7 Binding of Substrates to Adenylate Kinase .... 9

I I . THEORETICAL CONSIDERATIONS OF NMR TECHNIQUES ...... 14 Chemical Exchange Effect on NMR Line Shape .... 14 Determination of Stoichiometry by NMR Titration . . 17 Use of 170 NMR Linewidth to Determine Site of Coordination ...... 17 3 1 P(1 7 0) NMR Line Sharpening Effect in E*S co m p lex ...... 19

I I I . MATERIALS AND METHODS...... 23 Materials ...... 23 Purification of Solvents ...... 24 Ion Exchange Columns and Chromatographic Methods . 24 Adenylate Kinase Assay ...... 26 Sample Preparations ...... 27 Synthesis of Nucleotides ...... 30 NMR Methods ...... 37 Calculation of Stoichiometry ...... 43

v IV. THE INTERACTION OF M(II) WITH ADP AND ATP...... 45

31P NMR Properties of Alkaline Earth M(11) »ADP and M(II)-ATP ...... 45 170 NMR Properties of Alkaline Earth M(II) -ADP and M( 11) • ATP ...... 55 The Comparison of 170 NMR R Values and Stability Constants ...... 51 The Interaction of Cd(II) with A D P ...... 62 The Interaction of Zn(II), Hg(II) with A D P ...... 69 D iscu ssio n ...... 74

V. THE INTERACTION OF M(III) WITH A T P ...... 85

31P NMR Properties of M(III)-ATP Complexes ...... 85 170 NMR Properties of M(111)•ATP Complexes ...... 95 XH NMR Properties of M(III) -ATPComplexes ...... 105 D iscu ssio n ...... Ill

V I. 31P NMR STUDIES OF BINDING OF AMP AND ATP TO ADENYLATE KINASE ...... 117

The Stoichiometry of AK:ATP...... 117 The Crossbinding of AMP ...... 123 Differentiation Between the MgATP and AMP Sites of AK ...... 125 D iscu ssio n ...... 133

VII. SOME PRELIMINARY OBSERVATION ONAK-M*ATP ...... 143

31P NMR Experiments of AK*Sc(III)*ATP ...... 145 31P NMR Experiment ofAK-Cd( 11) -ATP ...... 154 31P NMR Experiment of AK»Mg(II)«ATP ...... 157

LIST OF REFERENCES ...... 161

APPENDIX...... 169

vi LIST OF TABLES

TABLE PAGE

1.1 Some physical properties for M(11) and M (III) ...... 6

4.1 31P NMR parameters of M(11) -ADP, M(11) -ATP complexes at pH 8.0 47

4.2 Nonlinear least square fitting results for stoichiometry of M(II)-ADP, M(II)-ATP ...... 49

4.3 Summary of 170 NMR results of M(11)-ADP, M(111)-ATP . . 59

4.4 Log K values of M( 11) -ADP and M( 11) • ATP ...... 61

5.1 31P NMR properties of M(III)-ATP complexes at pH 8 . 0 ...... 8 8

5.2 Nonlinear least square fitting results for stoichiometry of M(III) -ATP ...... 91

5.3 Summary of 170 NMR results of M( 111) -A T P ...... 100

5.4 Proton NMR results of M etal(III)•ATP complexes at pD 8 . 0 ...... 107

5.5 Ln(III)-triphosphate Distance ...... 113

6.1 31P NMR parameters of free and enzyme-bound substrates of AK at 10°C, pH 7.8 120

6.2 Nonlinear least square fitting results for stoichiometry of [AK]:[ATP] ...... 118

6.3 31P NMR results of AMP titrated with AK ...... 124

6.4 31P NMR chemical shifts of free and enzyme-bound nucleotides ...... 134

7.1 31P NMR parameters of free and AK bound ATP at 10°C, pH 7.8 147

7.2 AK kinetic results of Mg(II), Cd(I I ) complexed to A T P ...... 155

v ii LIST OF FIGURES

FIGURE PAGE

1.1 31P NMR spectra of MgATP free and bound to adenylate kinase, at pH 7.0 and T = 1 5 ° ...... 12

2.1 Effect of chemical exchange on line shape and resonance position ...... 16

3.1 31P NMR spectrum of [ 1 7 0 3 ]AMP...... 31

3.2 31P NMR spectrum of [a- 1 7 02]A D P ...... 32

3.3 31P NMR spectrum of [b- 1 7 0 3 ]ADP ...... 33

3.4 31P NMR spectrum of [a- 1 7 02]A T P ...... 34

3.5 31P NMR spectrum of [e- 1 7 02]ATP ...... 36

3.6 31P NMR spectrum of [y- 1 7 03]A T P ...... 37

3.7 Undecoupled and 3^-decoupled 170 NMR spectra of [y-1 7 03]ATP (10 mM, pH 8.0) 40

3.8 3IP NMR spectrum of TMP (15%) in CgD 6 ...... 42

4.1 31P NMR spectra of ADP (10 mM, pH 8.0) with varying concentrations of Ca(II) ...... 46

4.2 Titration curves of 31P chemical shifts of ADP (10 mM, pH 8.0) with CaCl2 (a), SrCl2 (b), and BaCl2 (c) 50

4.3 31P NMR spectra of ATP (10 mM, pH 8.0) with varying concentrations of Sr(II) ...... 53

4.4 Titration curves of 31P NMR chemical shifts of ATP with SrC12 ...... 54

4.5 170 NMR spectra (40.68 MHz) of [

4.6 170 NMR spectra (40.68 MHz) of [a - 17 0?]ATP, [B-1 7 02]ATP and [y- 1 7 03 ]ATP, and the corresponding complexes with alkaline earth metal ions ...... 60

v i i i Titration curves of *H NMR chemical shifts of ADP (10 mM in D2 0, pD 8.0) with Cd(II) ...... 64

Titration curves of 31P NMR chemical shifts of ADP (10 mM, pH 8.0) with Cd(II) ...... 66

Titration curves of 170 NMR line widths (a) and chemical shifts (b) of [a- 1 7 02]ADP and [e - 1 7 02]ADP with Cd(II) ...... 67

Titration curves of 31P NMR chemical shifts of ADP with Zn(II) ...... 70

Titration curves of NMR chemical shifts of ADP (10 mM in D2 0, pD 8.0) with Zn(II) ...... 71

Titration curves of 170 NMR line widths (a) and chemical shifts (b) of [o- 1 7 02]ADP and [8- 1 7 03]ADP with Zn(II) ...... 72

170 NMR spectra of [

!H NMR spectra of ADP titrated with varying concentrations of Hg(II) ...... 76

Proposed structure of the reactive CM2 (ATP)]2 (0H)“ dimer ...... 84

3lP NMR (81.0 MHz) spectra of ATP (10 mM, pH 8.0), with varying concentrations of ScCl 3 ...... 87

31P NMR spectra showing the assignment of the P signal of Sc( 111) (ATP) 2 ...... ,T . . . 89

31P NMR (81.0 MHz) spectra of ATP (10 mM, pH 8.0), with varying concentrations of LuC1 3 ...... 90

31P chemical shifts of ATP as a function of [Lu(III)]/[ATP] ...... 92

31P NMR (81.0 MHz) spectra of ATP (10 mM, pH 8.0), with varying concentrations of LaCl 3 ...... 93

31P chemical shifts of ATP as a function of [La(III)]/[ATP] ...... 94

170 NMR spectra (40.68 MHz) of [a -i 7 02]ATP (10 mM in 1 7 0-depleted water, pH 8.0) with varying concentrations of ScCl 3 ...... 96

ix 5.8 170 NMR spectra (40.68 MHz) of [b- 1 7 02]ATP with varying concentrations of SCCI 3 ...... 97

5.9 170 NMR spectra (40.68 MHz) of [y- 1 7 03]ATP with varying concentrations of SCCI 3 ...... 98

5.10 170 NMR spectra (40.68 MHz) of [y- 1 7 03]ATP (10 mM in 1 7 0-depleted water, pH 8.0) with varying concentrations of L a C ^ ...... 1 0 1

5.11 170 NMR line widths of [a- 1 7 02 ]ATP, [B-1 7 02]ATP and Cy-1 7 03]ATP (10 mM) with varying concentrations of LaCT 3 ...... 102

5.12 170 NMR chemical shifts of [a-1 7 02 ]ATP, [B-1 7 02]ATP and[Y- 1 7 0 3 ]ATP (10 mM) with varying concentrations of LaCT 3 ...... 103

5.13 *70 NMR spectra (40.68 MHz) of [a- 1 7 02]ATP (10 mM in 1 7 0-depleted water, pH 8.0) with varying concentrations of LuCl 3 ...... 104

5.14 *H NMR (200 MHz) spectra of ATP (20 mM in D 2 0, pD 8.0) with varying concentrations of ScCl 3 ...... 106

5.15 LH NMR chemical shifts of H3 , H2, and H o f ATP (20 mM, pD 8.0) with varying concentrations of LaCl 3 ...... 103

5.16 XH NMR chemical shifts of Hq , H2, and Hj' of ATP (20 mM, pD 8.0) with varying concentrations of L11CI3 ...... 109

5.17 The proposed structure of Sc(III)(ATP ) 2 ...... 115

6.1 PQ resonance of ATP bound to varying concentrations of AK ...... 119

6.2 31P spin-spin coupling constants J R, J R as a function of [AK]/[ATP] ...... 121

6.3 The linewidths of the P and P of ATP as a function of [AK]/[ATP] 122

6.4 3ip NMR spectra (81.0 MHz) of [ct- 1 7 02]ATP with varying concentrations of A K ...... 127

6.5 31P NMR spectra (81.0 MHz) of [ 1 7 03]AMP with varying concentrations of A K ...... 128

X 6 . 6 Simulated 31P NMR spectra of 170 labeled and regular AMP bound to A K ...... 130

6.7 31P NMR spectra of MgATP in the presence of AK as a function of tim e" ...... 132

6 . 8 Simplified representation of the crystal structure of A K ...... 139

6.9 Proposed structure of the MgATP site of AK ...... 140

7.1 31P NMR spectra (202.5 MHz) of Sc(III)(ATP ) 2 titrated with varying concentrations of AK ...... 146

7.2 31P NMR spectra of [y- 1 7 03 ]ATP:AK = 4:1 titrated with S c(III) ...... 149

7.3 31P NMR spectra of [y- 1 7 0 3 ]ATP titrated with AK . . . 153

7.4 31P NMR spectra of AK»ATP binary complex titrated with Cd(II) ...... 156

7.5 3IP NMR spectra of AK*ATP binary complex titrated with Mg(II) ...... 159

xi ABBREVIATIONS AND SYMBOLS

Abbreviations and symbols used in this dissertation are as fol 1 ows:

A, Adenine;

AdO, Adenosine;

ADP, Adenosine-5'-diphosphate;

AK, Adenylate kinase;

AMP, Adenosine-5"-monophosphate;

AppA, Diadenosine diphosphate;

5^-AdoP^, Adenosine-5'-tetraphosphate;

ATP, Adenosine-5'-triphosphate;

ATPas, Adenosine-5'-[l-thiotriphosphate];

ATPBs, Adenosine-S'-I^-thiotri phosphate];

CK, carbamate kinase;

DCC, N,N*-dicyclohexyl-carbodi imide

DMF, N,N-Dimethylformamide;

DMSO, Dimethyl sulfoxide;

DTE, Dithioerythritol;

EDTA, Ethylenediaminetetraacetate;

EPR, Electron paramagnetic resonance;

FID, Free induction decay;

eAMP, l,N6 -ethenoadenosine-5'-monophosphate;

gATP, l,N 6 -ethenoadenosine-5'-triphosphate;

Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid;

Kd, dissociation constant;

Km, Michael is constant;

x i i LDH, Lactate dehydrogenase;

Ln(III), Lanthanide ions;

M(II), divalent metal ions;

M(III), trivalent metal ions;

M.W., Molecular weight;

NAD+, Nicotinamide adenine dinucleotide;

NADH, Nicotinamide adenine dinucleotide, reduced form

NMR, Nuclear magnetic resonance;

NOE, Nuclear Overhauser effect;

0, Oxygen-17 isotope;

PEP, Phosphoenolpyruvate;

PGK, Phosphoglycerate kinase;

PK, Pyruvate kinase;

Rp, R configuration at chiral phosphorus center;

Sp, S configuration at chiral phosphorus center;

TEA, Triethanol amine;

TEAB, Triethyl ammonium bicarbonate;

TEA-HC1, Triethanol amine hydrochloride;

TLC, Thin layer chromatography;

Tp Spin-lattice (longitudinal) relaxation time;

Trizma, Tris(hydroxymethyl) aminomethane;

2D, Two dimensional;

UV, Ultraviolet. CHAPTER I

INTRODUCTION

This dissertation deals with the structural problems involved in the interaction of adenine nucleotides (ATP, ADP, and AMP) with divalent and trivalent metal ions and with adenylate kinase (AK).

Although these problems have been subjects of extensive research since the early 1960's, some of the fundamental issues such as mode of coordination, stoichiometry, etc., remain unresolved. The main thrust of this dissertation is to employ various NMR techniques to tackle some of the problems. In this chapter, I describe the background and define the specific problems.

1.1 The Interaction of M(II) with ADP and ATP

The enzyme-catalyzed phosphoryl transfer reactions are among the most fundamental processes in biochemistry. In many cases, the enzymes use magnesium-chelated ATP or ADP as a substrate. There are two structure-related questions which have not been resolved: (i) why does nature select Mg(II) for reactions involving adenine nucleotides in most kinases? (ii) how do the various divalent metal

ions differ in thei.r binding to nucleotides? In order to approach the answers to the questions above, a systematic study of various 2 divalent metal ion«nucleotide complexes is needed.

Following the pioneering work by Cohn & Hughes (1962), nuclear magnetic resonance spectroscopy has been applied during the past two

decades to study the structure of metal ion«nucleotide complexes. 3*P NMR chemical shifts were previously employed to deduce the interaction of metal ions with the phosphate moiety of nucleotides.

Unfortunately, the interpretation of these studies have been

controversial. For example, even the structure of the most

extensively studied MgATP is still not resolved conclusively. In a

31P NMR experiment, Cohn & Hughes (1962) reported that MgADP and

MgATP exist mainly as a, B-bidentate and 6 , y-bidentate,

respectively, based on the downfield chemical shift of the Pa, P^

signals of ADP, and the P^, PY signals of ATP. However, on the basis

of a similar 3*P NMR experiment, Kuntz & Swift (1973) suggested that

MgATP is a, 8 , y-tridentate. Tran-Dinh et al. (1975) have reported

that Mg(11) binds exclusively to the Pp of ATP, whereas Tran-Dinh &

Roux (1977) have postulated that Mg(II) binds exclusively to the Pa

of ADP. Gupta & Mildvan (1977) have argued that since the chemical

shift of the Pa of ATP behaves quite similarly to the PQ of ADP, and

since MgADP is believed to be an a, B-bidentate, the Mg(11) should

also interact with the a-phosphate of ATP. Ramirez & Marecek (1980)

suggested that MgATP is a mixture of o,B-, B,y-, and a,y-bidentates,

whereas Bishop et a l. (1981) suggested that MgATP exists

predominantly as a, B, y-tridentate. Q 1 The controversy in using P chemical shift method for determining these structures is best presented by Jaffe & Cohn (1978). In the31P NMR studies using phosphorothioate analogs of adenine nucleotides, the authors repeated their earlier work on the effect of added magnesium salts to ATP. They concluded that this method cannot be employed to specify the metal ion binding site.

Ramirez & Marecek (1980) further concluded that the effects of divalent metal ions on 3*P NMR shifts are the result of changes in the conformation of the polyphosphate chain, and therefore, bear no simple correlation to the particular phosphate group with which the metal cation is bound. The argument has been supported by recent reports in which 3*P NMR chemical shifts were shown to be quite sensitive to changes in both O-P-O bond angles and P-0 torsional angles (Gorestein; 1981, 1984). As a result, the determination of charge neutralization sites (Jaffe & Cohn, 1978., Gerlt et a l., 1982) and metal coordination sites by the change of 3*P NMR chemical shifts are questionable.

A possible alternative to the 31P chemical shift method for studying metal ion»nucleotide complexes is to use the *70 NMR line broadening effect. The site specificity of diamagnetic metal ions binding on nucleotides has been suggested by observing the 170 NMR line widths of substitution-inert Co(111) complexes of * 7 0-labeled

ADP and ATP (Huang & Tsai, 1982). Since the binding modes of CoADP and CoATP have been identified by X-ray (Cornelius et a l., 1977,

Cleland, 1982), these compounds are excellent models to show the binding site specif icity. The interaction of Mg(11) with ATP has also been demonstrated by this *70 NMR line broadening method (Huang

& Tsai, 1982), and it was thought to exist predominantly as an 4 equilibrium mixture of a, f?, y-tridentate (50 - 60%) and g, y-

bidentate (40 - 50%) (Huang & Tsai, 1982) (see scheme 1.1). The

result was independently supported by NMR chemical shift calculations (Pecoraro et al., 1984).

0 0 0 0 0 I 0-P-0- M-P-OAd O-P-O-P-O-P-OAd I I °N 9 K P 0 NsMg'(H2 0 ) 3 %‘Mg(H2 0) 4

Scheme 1.1

*H NMR chemical shifts have also been extensively used to observe the structures of the base ring moiety of the metal

1on»nucleotide complexes. Cohn & Hughes (1962) have shown an effect of Zn(II), but not of Mg(II) and Ca(II), on the chemical shift of Hg on the adenine ring. The interpretation was that Zn(II) directly coordinates the N 7 of the adenine ring. Based on the chemical shift changes of the ring protons, Granot & Fiat (1977) have suggested the formation of Co(ATP ) 2 and Ni(ATP ) 2 ternary complexes. Fan et al.

(1977) observed the concentration dependence of the Hg, H 2 and Hj' chemical shifts and concluded that cyclic AMP and ATP self associate at higher concentration ranges (>0.03M). Similar experiments and results have been reported (Lam & Kotowycz, 1977, Mitchell & Si gel,

1978., Scheller et al. 1981., Scheller & Sigel, 1983). Bock (1980) has observed that Zn(II), Cd(II), and Ag(I) induce a downfield shift of the Hg resonance along with an upfield shift of the H2 signal. 5

The effect on Hg was interpreted as the direct metal ion coordination to N7 , whereas base stacking affected the H2 resonance. Recently, Hg chemical shift variations were employed to study the structure of the

A l(III)‘ATP complex (Karik et al. 1983). In summary, if complex formation involves a stacking of adenine rings, then upfield shifts of these ring protons should be observed due to ring current effects of one molecule stacked on the other (Helene et a l., 1971, Wagner &

Lawaczeck, 1972). If metal ion coordinates to the base ring, downfield shift of ring protons are expected (Cohn & Hughes, 1962).

Compared to the extensively studied MgATP and MgADP, the coordination of other alkaline earth metal ions to the adenine nucletides has drawn less attention. Reported in Chapter IV is a systematic *H, ^ 0 and 3*P NMR observation of alkaline earth metal ions complexed with ADP and ATP. The research also includes a study of group 2B (Zn(II), Cd(II), Hg(II)) for a parallel comparison.

Table 1,1 lists some physical properties for these M(II) ions.

The purpose of this study is to determine stoichiometry of metal ion*nucleotide complexes by 3*P NMR titra tio n curves; metal ion cordination sites by ^ 0 NMR; and to determine the presence of self stacking by *H NMR. By interpreting our results in light of those that have been previously reported, I conclude that alkaline earth metal ions form 1 : 1 complexes to nucleotides interacting with the phosphate chain in the order Mg(II) > Ca(II) > Sr(II) > Ba(II),

Zn(II) and Cd(II) form complexes with mixed stoichiometries, such as

1:1, 1:2, 2:2. The observed base ring stacking is caused by the metal ions interacting with N 7 of base ring. 6

Table 1.1 Some Physical Properties for M(11) and M(111)a

Electronic E°(V) for Ionic Charge

Element Configuration M+ 2 +2e" ^ Ms Radii(A) radius

M+3 +3e“ *=* Ms

Mg [Ne]2s 2 -2.37 0.78 3.1

Ca [Ar]3s2 -2.87 1.06 2 . 0

Sr [Kr]4s2 -2.89 1.27 1 . 8

Ba [Xe]5s2 -2.90 1.43 1.5

Zn [Ar]3d^4s 2 -0.76 0.69 2.9

Cd [Kr]4d 1 0 5s2 -0.40 0.92 2 . 2

Hg [Xe]5d 1 0 6s2 0.85 0.93 2 . 1

Sc [Aritfd^s 2 - 1 . 8 8 0 . 6 8 4.4

La [X e^d ^s 2 -2.52 1.06 2.9

Lu [Xe]4f1 4 5d 1 6s2 -2.25 0.85 3.5

a0btained from Cotton (1980). 7

1.2 The Interaction of M (III) with ATP

Many biochemically active divalent metal ions, such as Mg(II),

Ca(II), and Zn(II) are spectroscopically silent. In such cases, it

is often possible to substitute an ion with useful spectroscopic

characteristics for the native diamagnetic metal ion. The

replacement of Mg(11) and Ca(II) by the trivalent lanthanide ions,

Ln(III), in the study of metal ion«nucleotide complexes and their

interaction with enzymes are typical examples of this approach (for a

recent review, see Horrocks, 1982).

Ln(III)*ATP complexes can be effectively employed to probe the

structure of MgATP binding on the enzyme only after its structural

and thermodynamic properties have been solved. With the assumption

of 1:1 Ln(III)•ATP complexes, the dissociation constants have been

studied by various techniques such as potentiometry (Galea et a l.,

1978), spectrophotometry (Ellis & Morrison, 1974) and enzyme kinetics

(Morrison & Cl el and, 1980; Viola et al., 1980; Morrison & Cl el and,

1983). Tanswell et al. (1975) have suggested that Ln(III) coordinate

to Pp, Py of ATP based on Ln(III)-induced *H and 31P NMR shifts

assuming a 1:1 stoichiometry. This stoichiometry has also been

proposed in the kinetic experiments of Ln(111) -ATP binding to

hexokinase, the dissociation constants of these complexes were then

determined (Morrison & Cleland, 1983). The authors concluded that

the coordination is B, y-bidentate or a, B, y-tridentate depending on

the ionic radius of Ln(III). On the other hand, an EPR spin-echo method has been employed (Shimizu et a l. 1979., Shimizu et al. 1983) to probe ATP complexes of Ce(III), Nd(III), Er(III), and Yb(III). 8

The titration data suggested the formation of 1:2 and possibly 1:3

Ln(III)»ATP complexes of the metal ions with ATP. Recently, by utilizing the ability of Eu(III) to luminesce, of Pr(III) to act as a

^ P NMR chemical shift reagent, and of Gd(III) to enhance magnetic

resonance relaxation of nearby nuclei, Eads et al. (1984) have characterized the Ln(111) -ATP complexes. These results are also consistent with formation of a 1:2 Ln(III)-ATP complex at millimolar

ATP concentrations.

Due to the ambiguity concerning the coordination sites and the stoichiometry of M(111) *ATP complexes, it is worthwhile to further

investigate these problems. Compared to the extensively studied paramagnetic M(III) -ATP complexes (Shimizu et a l., 1979, Shimizu et a l., 1983, Tanswell et a l., 1975, Eads et a l., 1984), the effects of diamagnetic M(III) on nucleotides have been less studied. In

addition, one great advantage for observing such a system is that the presence of these diamagnetic metal ions at NMR concentratioins (~10 mM) cause no broadening the signals of nucleotides. The three trivalent diamagnetic metal ions chosen in this researh are La(111),

Lu(III), and Sc(111). The ionic radii and other properties, are also listed in Table 1.1. The two lanthanide ions chosen are diamagnetic, but have sufficient influence on the NMR resonances of the nucleotides to deduce the structure of these Ln(III)-ATP complexes.

By choosing Sc(111), with ionic radius 0.68A, I hoped to test the hypothesis of Morrison & Cleland (1983) that M(III) ions possessing ionic radii smaller than 0.88A coordinate to the R.y-phosphates of

ATP, whereas those with ionic radii greater than 0.88A appear as a, 9

B, y-tridentate.

In Chapter V, a systematic study of the complexes of ATP coordinating to these diamagnetic triv a le n t metal ions is performed using the multinuclear (*H, and ^ 0 ) NMR technique developed in

Chapter IV. The stoichiometry as well as the binding sites of these

N(III)*A TP complexes are discussed.

1.3 Binding of Substrates to Adenylate Kinase

Kinases catalyze reactions involving transfer of the y- phosphoryl groups of nucleotides to various nucleophilic acceptors

(except water) such as arginine, creatine, AMP, 3-phosphoglycerate, pyruvate, etc. (Walsh, 1979). Adenylate kinase (E.C.2.7.4.3), also known as "myokinase", and "ATP:AMP phosphotransferase", is important in maintaining the equilibrium among the adenine nucleotide pool

(Noda, 1973). AK catalyzes the reaction:

NgATP + AMP MgADP + ADP

The Y-phosphate of MgATP is transferred to the phosphate acceptor AMP through a direct phosphoryl transfer mechanism (Richard & Frey,

1978). The enzyme has been purified from a wide variety of sources which include the muscle of procine (Heil et a l., 1974), rabbit (Noda

& Kuby, 1957, a.b), calf (Kuby et a l., 1978), carp (Noda et a l.,

1975), human (Thuma et a l., 1972; Kuby et a l., 1982), rat (Tamura et a l., 1980), bovine liver mitochondria (Markland & Wadkins, 1966 a.b.), human liver mitochondria (Kuby et a l., 1982), human 10 erythrocyte (Tsuboi & Chervenka, 1975), rat brain (Pradhan & Criss,

1976), and baker's yeast (Chin et a l., 1967). The enzyme from porcine muscle has a single polypeptide chain with M.W. of 21,500.

The amino acid sequence (Heil et a l., 1974) and the x-ray crystal structure have also been deduced (Schulz et a l., 1974; Sachsenheimer

& Schulz, 1977; Pai et a l., 1977). The enzyme from rabbit muscle is highly homologous to the porcine enzyme (Kuby et a l., 1984), and has also been studied extensively.

Due to the low M.W., this enzyme appears to be one of the most suitable proteins for NMR study of the structure and function of phosphoryl transfer enzymes. The interaction of AK with substrates has been studied by 31P (Nageswara Rao & Cohn, 1977b; Nageswara Rao et a l., 1978; Vasavada et a l., 1984) and NMR (Price et al., 1973;

Smith & Mildvan, 1982; McDonald et a l., 1975; Kalbitzer et a l., 1982;

Rosch & Gross, 1984; Fry et a l., 1985). Kinetic constants have been

O 1 obtained by saturation or inversion transfer of P spins (Brown et a l., 1977; Brown & Ogawa, 1977), and by 2-D NMR cross-relaxation on exchange experiments (Mendz et a l., 1986).

Kinetic studies (Rhoads & Lowenstein, 1968; Hamada & Kuby, 1978) have suggested two distinct substrate binding sites in the enzyme from rabbit muscle: the MgATP site, which binds magnesium-chelated

ATP and ADP, and the AMP site, which binds unchelated AMP and ADP.

By differentiating the bound ADP and MgADP, the asymmetry of the Ol binding sites of porcine enzyme was further supported by P NMR studies (Nageswara Rao et a l., 1978; Nageswara Rao & Cohn, 1977b;

Vasavada et a l., 1984). Two proteolytic fragments, corresponding to 11 residues 1-44 and 172-194 of rabbit muscle AK, have been shown to bind MgeATP (l,N6-ethenoadenosine 5'-triphosphate) and eAMP, respectively (Hamada et a l., 1979). Recently, *H NMR NOE technique has also demonstrated the selective binding of Cr(III)ATP complex to a synthetic peptide corresponding to the residues 1-44 of rabbit muscle AK (Fry et al., 1985).

It would appear that the substrate binding sites of this enzyme have been well defined. However, the cross binding of substrates

(i.e. ATP binding to the AMP site and vice versa) in native AK is still unclear. The possibility that AMP may bind to the MgATP site has been suggested by several studies. Rhoads & Lowenstein (1968) observed that the rabbit muscle adenylate kinase reaction rate is inhibited by a high concentration of AMP and interpreted the result as the existence of AK*AMP*AMP complex. By observing ultraviolet difference spectra of nucleotides incubated with yeast AK, Tomasselli

& Noda (1983) suggested that AMP binds at the MgATP site in the absence of cosubstrate. Binding of ATP to the AMP site has also been proposed by x-ray crystallography on porcine AK (Pai et a l., 1977), and by an equilibrium binding study on rabbit muscle AK (Kuby et al.,

1962). Nageswara Rao et al. (1978) proposed that ATP can bind to both the AMP and the MgATP sites of porcine AK based on the ^ P nmr experiment shown in Figure 1.1,D. The upfield Pp resonance (21.0 ppm) was assisnged as ATP bound to the AMP site. The downfield P 0 resonance (17.7 ppm) was assigned to the MgATP binding at the MgATP site.

Not only the binding site, but also the stoichiometry of the E»S 12

« -p r-P

p.P

■vrtT^A/^ '1,M^VwA/v-v>

CHEMICAL SHIFT (p p m )

Figure 1.1 NMR spectra of MgATP complex, free and bound, to porcine or carp adenyalte kinase, at pH 7.0 and T = 15°. A, free

Mg«ATP (ATP, 15.1 mM; MgCl 2 » 17.5 mM); potassium Hepes, 50 mM. B,

Mg»ATP (ATP, 3.7 mM; MgC^, 3.9 mM) bound to porcine adenylate kinase, 3.9 mM; potassium Hepes, 100 mM. C, Mg*ATP (ATP, 1.9 mM;

Mg(CH3 C0 0 )2 » 2.0 mM) bound to carp adenylate kinase, 2.3 mM; potassium Hepes, 100 mM, D, Mg«ATP bound to porcine adenylate kinase with excess Mg(II). (enzyme, 2.7 mM; ATP, 2.5 mM; MgC^, 5,8 mM; potassium Hepes, 100 mM). (Adapted from Nageswara Rao & Cohn, 1978.) 13 complexes of AK is not known conclusively. The binding of ATP to both sites (Figure 1.1,D) implies a stoichiometry of 2 for ATP binding to porcine AK. The equilibrium binding experiment by Kuby et al. (1962) revealed a stoichiometry of 1.8 for both ATP and MgATP bound to rabbit enzyme. On the other hand, 1:1 stoichiometry of

[AK]:[ATPJ were also suggested by several studies. Nuclear magnetic relaxation and electron paramagnetic resonance techniques have been used by Price et al. (1973) to examine the binding of substrates to porcine muscle adenylate kinase. The proton relaxation rate showed that there is one binding site for MnATP or ATP per mole of enzyme.

In the reports on the binding studies of l,N^-etheno analogs of the adenine nucleotides on rabbit and calf muscle adenylate kinase by fluorescence quenching and UV difference spectrum, 1:1 stoichiometry were also suggested (Hamada et al., 1979). In a later equilibrium dialysis binding study, Tomasselli & Noda (1980) found mitochondria

AK from beef heart to bind MgATP or ATP with 1:1 ratio.

The controversy regarding the cross binding of substrates and the stoichiometry of [AK]:[ATP], will be the subject of the investigation in Chapter VI. 3*P NMR titration of ATP with AK indicates a stiochiometry of 1:1. The 3 *P(* 7 0) NMR property of [a-

1 7 02 ]ATP*AK binary complex is different from that of [^ 7 03 ]AMP*AK binary complex, which suggests different binding sites of AMP and ATP in AK. A broadened AMP signal at the ratio of [AMP]:[AK] = 1:0.2 implies the cross binding of AMP at the MgATP site. CHAPTER II

THEORETICAL CONSIDERATIONS OF NMR TECHNIQUES

Several NMR techniques have been employed in this dissertation to study the structures of metal ion*nucleotide complexes and the binding of nucleotides to adenylate kinase. In the following sections, the theoretical basis of these methods is provided.

2.1 Chemical Exchange Effect on NMR Line Shape

The effect of chemical exchange on the line shape of a resonance is important to the interpretation of NMR data. Detailed theories and the calculation of these line shapes in simple spin systems are available (Kaplan & Fraenkel, 1980). Only a few limiting conditions relevant to this research are briefly discussed below.

If wA and ug are two resonance frequencies, and AP 0 (A) and

AP0 (B) represent their corresponding line widths in the absence of exchanges in a two-site exchange process, three limiting conditions are of interest (Figure 2.1):

|wA - wg| » t a _ 1 , T g "1 (slow exchange)

jo)A - ug.J s t a _ 1 , tg " 1 (intermediate exchange)

|wA - Ug| << ta_1, Tg " 1 (fast exchange)

14 15 where rA and tb are the lifetimes in the two states during exchange.

Under slow exchange conditions, the two resonances will s till be centered around

AP(A) - aPq(A) = t a - 1

AP(B) - APq(B) = t b - 1

Under fast exchange conditions, a single resonance is observed at an intermediate frequency (to) with intermediate line width (AP) given by

w = PA“A + PBWB Ap = paapo (a ) + pbapq (b)

where PA and Pg are the fractional populations ofthe exchanging species. If the intermediate exchange conditions are obtained, both frequency shift and line broadening result.

It must be noted that the above is a simplified description of the exchange effects and does not include, for example, effect of spin-spin coupling on the line shape. For the purpose of understanding the results considered in this dissertation, this description is adequate. 16

No ♦- A . Exchange

w .

T"l T_l a b ^ TA , r B Slow A w “ - ^ ( A a/ *" A (i/rt ) / 2 = r " * 0 A

Intermediate OJ. - Cl) ~ r ' 1 r _l A B ~ A , B

Fast W ,-W n A B

y v.

Figure 2.1 Effect of chemical exchange on line shape and resonance position. 17

2.2 Determination of Stoichiometry by NMR Titration

In principle, if there are changes in chemical shifts (or in any other NMR parameters) of nucleotides due to the formation of complexes with metal ions or with enzymes, the stoichiometry can be deduced for either fast or slow exchange conditions. For fast exchange, the stoichiometry can be determined from the changes of the chemical shifts (or the relevant parameters) of nucleotides with concentration variation of metal ions or enzymes. For the slow exchange condition, the stoichiometry can be determined from the

relative peak intensities of free and bound species.

NMR chemical shift change of a nucleotide is expected to be a function of the change of the phosphate chemical environment. A variety of factors, such as pH, metal chelation, can result in such chemical shift changes (Cohn & Nageswara Rao, 1979). In Chapter IV and V, the stoichiometry of metal»nucleotide complexes is mainly determined by the detailed observation of the changes in ^ P NMR chemical shifts of the titration courses of adeneine nucleotides with metal ions. The stoichiometry of ATP bound to adenylate kinase, as discussed in Chapter VI, is also investigated by ^*P NMR titration experiments. Although the ^^P chemical shifts of ATP are unaffected by the binding of AK (Nageswara Rao et al; 1978), the changes in other parameters (Jap, and linewidths) during the titration course are used to determine the stoichiometry.

2.3 Use of the *^0 NMR Linewidth to Determine Site of Coordination

Huang & Tsai (1982) have defined the R values as a measure of 18

the “line broadening effect" for metal 1 on«nucleotide complexes in

170 NMR: AO.-ACL n _ b f R M f

Where AOf and AOjj represent the ^70 line widths of free and bound nucleotides, respectively. Upon titration of * 7 0-labeled ATP with

Mg(II), the R values has been shown to increase linearly up to

[Mg(II)]/[ATP] = 1.0 (Huang & Tsai, 1982). By utilizing the separate dlastereomers of Co(NH 3 )4 ADP (ADP is chirally labeled with *70 at the

Pa of ADP), Sammons et a l., (1985) have further established that the positive R values represent the site specificity of metal ion coordination. The theoretical basis of *70 NMR line width is as follows. A nucleus with nuclear spin I greater than 1/2 possesses an electric quadrupole moment eQ. The dominant relaxation mechanism for quadrupolar nuclei comes from Interactions of eQ with an electric field gradient eq at the nucleus and the modulation of these interactions by rotational motion (James, 1975). In the extreme narrowing conditions, I.e., very fast molecular motions with respect to resonance frequency, which is the case for small molecules in solution, the contribution of nuclear quadrupole relaxation to the relaxation rate can be expressed as:

1 1 1 3 2I + 3/1a n^we^qQ^ (on 9 7“ ~ T— ~ T — ~m~2------{1 + - j)(—p) t leq. d.i) 'q . 1 lq 2 q 4U T ( 2 I- 1 ) J " r

O where e qQ/h 1s the nuclear quadrupole coupling constant (NQCC), n is the asymmetry parameter, and tp the rotational correlation time 19

(Abragam, 1961). *70 nuclei possessing I = 5/2 labeled in nucleotides are satisfied with eq. 2 . 1 .

Since is generally smaller than 3 msec for biophosphates, the approximation AO l/*Tq can be justified, where AO is the linewidth of 170 signals. Equation (2.1) becomes:

A0 ( 1 + -j“)(NQcc)^Tr (eq. 2 . 2 )

Thus, the linewidth AO is directly related to n» NQCC, and xr. Since

0 < n < 1, the effect of (1 + n 2 /3) is not more than 33%. However, the "symmetry" can affect both n and NQCC, and the effect of the latter can be quite large. On the other hand, if NQCC can be kept constant, the * 7 0 linewidth can very well reflect changes in xr .

The rotational correlation time xp is related to the molecular radius (a), the viscosity of the medium ("n) and the temperature (T) according to the Debye-Stokes theory (Abragam, 1961):

xp = 4na 3 n/3kT (eq. 2.3)

where k is the Boltzmann constant. The AO of ATP follows the order a -^ 0 > B-*70 > y- ^ 0 and that of ADP follows the order a-*70 >

B-* 7 0, which may explained the larger restriciton in rotational motions for Inner phosphates.

2.4 31P(170) NMR Line Sharpening Effect in E»S Complex

For small biophosphate molecules, if th e ^lp nucleus is bonded 20

directly to the quadrupolar * 7 0 nucleus, at the extreme narrowing

limit condition (w2tc2<<1), the following simplified equations hold

(Bruzik & Tsai, 1984):

Tlsc

—L_ ~ liL Tr2j2T (ecl* 2,/*) t2 s c ~ 3 q

where 1 /T^sc and 1 /T2sc are the contribution of scalar relaxation to the longitudinal, transverse relaxations, J is the 31p_17g spin-spin

Q 1 coupling constant. Under this condition, 1/T 2 s 1/T2sc for J iP, and

Ti s T2 Z Tq for * 7 0, which ju stifies the approximations for AO (the linewidth of the *70 NMR line width), and APSC (the contribution of scalar relaxation to the linewidth of 3*p NMR resonance) as :

A0=l/tr Tq

‘'’sc”1/’ T2 sc

Thus, eq. 2.4 becomes

APscA0=35/3 J 2 (eq. 2.5)

In E»S complexes, if the enzyme is small or the phosphoryl group at the active site .has some freedom of internal rotation, the extreme narrowing limit is s till valid. If the rotational correlation time,

Tr, of 170 increases due to substrate binding to the enzyme, Tq 21 should decrease, (i.e. AO increases) and the effect of scalar relaxation on the linewidth of 3*P should diminish (i.e.

APS(.decreases). According to Equation 2.5, the ^P (^O ) signa] should “sharpen" to some extent.

Such 31p(170, nmr "line sharpening" effects have been observed previously in ATP upon binding with diamagnetic metal ions (Sammons et a l., 1983., Shyy et a l., 1985), and in ADP upon binding to ribonuclease A (Tsai, 1982). However, two other cases which are shown as follow are still needed to be taken into consideration.

An intermediate situation which may occur is that if xr

p 3 increases by 1 0 - 1 0 , the extreme narrowing approximation may still be valid. In such a case, AO may increase from 500 Hz to 50-500 KHz, which is too broad to be detected. On the other hand, AP may decrease from 500 Hz to 5-0.5 Hz, and the 3 *p-170 signal would not be distinguished from the 3 1 p- ^ 0 signal, as in the case of HgP^OOg in glycerol.

On the other hand, if in E»S complexes, xp increases by a factor of 1 0 ^ or more (e.g., from 1 0 " 1 1 to 1 0 " 7 sec), the approximations

<< 1 may not apply. In this "nonextreme narrowing" condition, the 1 7 0 relaxation must be described by a sum of three decaying O I 1 *7 exponentials (Abragam, 1961), and the J1 p - A/o interaction can be even Ol more complicated. It is quite possible that the P relaxation will be dominated by the dipolar relaxation due to * 7 0 , which also results Ol in the broadening of the P NMR signal.

The 3 1 P(1 7 0) NMR method is used in Chapter VI to study the binding of AMP and ATP to adenylate kinase. Different 3 *P(* 7 0) spectral properties of AK«[^ 0 3 ]AMP and AK»[a-^U2]ATP suggest that these two nucleotides belong to different 3 1 P(1 7 0) range (i.e. a dramatic difference of xr ). Thus, the experiemnts support that AMP and ATP bind to different sites. CHAPTER I I I

MATERIALS AND METHODS

3.1 Materials

The following reagent grade chemicals were purchased from

Aldrich Chemical Company: tri-n-butylamine, phosphorus pentachloride

(PCI5 )» phosphorus oxychloride (P0C13), phosphorus pentoxide (PgOs)* tri-n-octylamine, triethyl phosphate ((EtO^PO), pyridine, triethyl amine, N,N-dimethylformamide (DMF), dimethyl sulfoxide

(DMSO), 1,4-dioxane, diphenyl chlorophosphate, potasium cyanate

(KOCN), and morpholine.

Puratronic grade metal oxide: ScgOg, La 2 0 3 » LU2 O3 ; metal chloride: CaC^* CdCl 2 » SrC^. BaC^* HgCl2 » MgC^; MgSO^, and

Zn(NO3 ) 2 were obtained from Alfa.

Hepes, Trizma hydrochloride, triethanol amine hydrochloride, ethylenediaminetetraacetate (EDTA), phosphoenolpyruvate (PEP), dithioerythritol (DTE), adenosine, AMP, ADP, ATP, ADP-morpholidate, diadenosine diphosphate (AppA), NADH, carbamate kinase, pyruvate kinase, lactate dehydrogenase, and 4-morpholine-N,N'dicyclocarboxami- dine were obtained from Sigma.

DEAE Sephadex.A-25 anion exchanger and Sp Sephadex C-25 cation exchanger were purchased from Pharmacia. Dicyclohexyl-carbodiimide

23 24

(DCC) and tributyl amine were obtained from Eastman. H2170 (51.0 atom % 1 7 0, 38.6 atom % 1 8 0) was obtained from Monsanto. 1 7 0- depleted water (0.00338 atom % 1 7 0) was obtained from Yeda Stable

Isotopes. All other chemicals were reagent grade.

3.2 Purification of Solvents

Generally, solvents were purified, dried and stored based on the procedures in "Purification of Laboratory Chemicals" (Perrin &

Perrin, 1966).

The following solvents were initially dried by refluxing with

CaH2 followed by distillation: pyridine, 1,4-dioxane, t-butyl alcohol, DMSO (dimethyl sulfoxide) and triethyl phosphate. Tri-n- butylamine, tri-n-octylamine and DMF (N,N-dimethylformamide) were stirred with CaH2 followed by vacuum distillation. Triethylamine was purified by distillation.

Anhydrous solvents were usually stored over 4A Linde molecular seives (Davison), which had been dried at 105°C overnight. The containers were capped with rubber heads or glass stoppers, sealed with teflon tape and stored in a dessicator.

3.3 Ion Exchange Columns and Chromatographic Methods

Cation Exchanger

(Dowex 50W-X8) Dowex 50W-X8 was employed to determine the concentrations of metal ion stock solutions and to convert nucleotides to pyridinium salts.

A 2 x 25 cm column of dry resin was washed with distilled water, 25

acid and base in the following order: 700 ml H 2 0, 700 ml IN NaOH,

700 ml H2 0, 700 ml IN HC1, and 700 ml HgO, (pyridinium hydrochloride

if needed). The residual Cl" ions were always checked with AgN 0 3 .

The H+ form of resin could be regenerated by washing with IN HC1

followed by distilled water.

Sp-Sephadex C-25 This cation exchanger was used to convert

nucleotides to sodium salts.

Dry resin was swollen in 1.0 M NaCl solution for several days.

Columns were packed by pouring the swelled resin into a 1 x 25 cm

column. This slurry was then washed with 1 £ of IN NaCl followed by

1 £ of distilled water. The final water eluent was also tested with

AgNOg for residual Cl". Regenerating the Na+ form of this resin was

achieved by washing with 1 £ of IN NaCl.

Chelex-100 Chelex-100 (Bio-Rad) was washed with distilled water

and packed in a 0 . 6 x 2 cm minicolumn.

Anion Exchanger (DEAE Spehadex A-25) Nucleotides were purified

with DEAE Spehadex A-25 anion exchanger. The dry resin was first

swollen in 1.0 M TEAB (Triethyl ammonium bicarbonate) buffer for

several days at 4°C. The resin was packed by pouring the slurry into the column (2 x 30 cm) and washed with 1.5 £ of 1M NaHC0 3 , 1.5 £ of

distilled water and 1.5 £ of the lowest concentration of TEAB to be

used in the linear gradient chromatography.

The buffer, TEAB, was prepared at 4°C in the following manner:

Distilled water was added to 550 ml of triethyl amine to make the total volume 4.0 £ ( 4 £ 1M TEAB). C02 was generated by placing

solid COg in a sealed suction flask. The gas produced was delivered 26 to the amine solution by means of a hose, connected to an aerator.

Meanwhile, a magnetic bar was kept stirring in the solvent bottle.

Usually, it took few hours for the buffer to be saturated, and that is judged by measuring a pH below 7.6.The stock solution wasstored at 5°C to prohibit C02 from escaping.

Linear gradients were formed by adding an equal volume of two different concentrations of buffer into two 2 £ bottles. The buffer flowed from the higher concentration bottle to the lower concentration bottle and the gradient concentration flowed from the lower concentration bottle to the column. A magnetic bar was used in the bottle containing the lower concentration. The elution profile was monitored by UV at

259 nm.

Thin Layer Chromatography In this study, TLC was routinely used to detect adenosine nucleotides. The developing solvent system was n-propanol, NH 4 OH and H20 with the ratio 6:3:1 by volume. The plates were coated with silica gel containing a fluorescent indicator

(Kodak). The Rf values for AMP, ADP and ATP were 0.4, 0.2, and 0.1, respectively.

3.4 Adenylate Kinase (Myoklnase) Assay

The activity of the porcine AK was determined using two coupled enzymatic reactions, pyruvate kinase and lactate dehydrogenase (Price et a l., 1973), according to the overall reaction shown below:

AK MgATP + AMP ^===^ ADP + Mg ADP

PK Phosphoenol pyruvate + MgADP » MgATP + pyruvate 27

LDH pyruvate + NADH + H+ NAD + lactate

The assay solution was composed of 0.1 ml of each of the following stock solutions: 1.2 mM ATP, 1.4 mM AMP, 0.1 mM NADH, 10.0 mM MgCl2, 1*0 mM PEP, and 0.5 ml of 0.15 M triethanol amine«HCl buffer containing 0.13 M KC1 and 3.0 mM DTE, pH 7.8 (Bergmeyer, 1980). 5 yl each of hexokinase and lactate dehydrogenase stock solutions (Sigma) were then added and monitored at 340 nm to ensure a flat baseline.

Lastly, AK was added, followed by the measurement of rate as the decrease in A-j^g, which was due to the formation of NAD+.

The concentration of AK was measured spectrophotometrically at

280 nm, with the extinction coefficient 5.4 for 1% solution (Schirmer et a l., 1970) and molecular weight of 21,500 (Kuby et a l., 1978).

The specific activity of the porcine AK, in general, was 1500 to

1800 units/mg.

3.5 Sample Preparations

Stock Solutions of Metal Ions The puratronic grade metal oxides

Sc2 0 3 » La2 03, and Lu 2 03 (99.99% from Alfa) were first dissolved in a minimum volume of 12 N HC1 upon gentle heating. The samples were then diluted with triple distilled water and rotary evaporated several times to remove excess HC1. After redissolving in triple distilled water, the concentrations of the solutions were determined by passing an aliquot through a Dowex 50W - X 8 (H+ form) column. 100 ml of eluent was usually collected in a flask, followed by titrating the released H+ ions with standardized NaOH. The stock solutions were then stored in plastic bottles, sealed with parafilm, and kept 28 at -20°C.

The stock solutions of M( 11) Cl 2 ancJ Zn(N 0 3 ) 2 were also prepared according to the procedure described above, except that M( 1 1 )Cl 2 anc*

ZnfNOg^ were dissolved in triple distilled water directly.

Stock Solutions of Nucleotides Nucleotides were purified and converted to their sodium salts by the following procedure: Crude samples were first applied to a DEAE-Sephadex A-25 column, which had been equilibrated with TEAB buffer. The column was eluted by gradient TEAB buffer. The fractions were monitored by UV at 259 nm and identified by checking with TLC. Peak fractions were pooled, and

TEAB buffer was removed by rotary evaporation. The nucleotides were then rotary evaporated twice with MeOH to remove the buffer salts.

The residue was dissolved in a minimum amount of triple distilled water and reapplied to a Sp-Sephadex C-25 (Na+ form) column.

Nucleotides were then eluted from the column with distilled water.

Peak fractions were collected and concentrated.

To remove trace contaminating metal ions, the sample solutions were usually passed through a 0.6 x 3 cm column of Chelex-100 (Bio-

Rad). The final solution was titrated to pH 7.8 with puratronic grade NaOH (Alfa), followed by quantitation (a molar extinction coefficient of 15,400 at 259 nm for adenine nucleotides), and kept frozen to avoid decomposition.

In general, ATP and ADP stock solutions were stable for at least one month. However, TLC examination of purity was performed before every experiment.

Enzyme Solutions The porcine AK was purified by J. Hart of this 29 laboratory according to the procedure of Kress et al. (1966) and stored as pellets in a holding solution containing 3.2 M (NH^) 2 S04, 1 mM EDTA, and 5 mM DTE, pH 6.0, at 4°C. The pellets were redissolved with a minimum volume of 0.07 M TEA-HC1 buffer containing 65 mM KC1 and 1.5 mM DTE, pH 7.8. The dissolved enzyme solution was then dialyzed in a dialyzing tube (Spectrum Med. Ind., M.W. cut off 4,000) against 10 mM EDTA, pH 7.8 (300 ml in 4 portions) followed by dialysis against the same TEA-HC1 buffer (300 ml in 3 portions). The enzyme solution was then concentrated in a Minicon-B15 protein concentrator. The final concentrations were usually ca. 2 mM.

Preparation of NMR Samples Samples in the study of metal»nucleotide interactions were prepared by mixing desired amounts of M(11) or M(III) with ADP or ATP stock solutions in ^ -d ep lete d water (for 170 NMR), in H 2 0/D20 (4:1 v/’v, for 31P NMR), or in 99.8%

D20 (for *H NMR). The pH was then adjusted to 8.0 (direct reading from pH meter) with NaOH/HCl or NaOD/DCl. In *70 NMR, the titrations were usually begun with spectra of free nucleotides followed by successive addition of metal ions (pH was readjusted, a new sample was prepared if ATP decomposition occured during the course of titration). For 3*P and ^H NMR, multiple samples of different ratios of metal ion: nucleotide were prepared. The samples for *H NMR were further lyophilized and redissolved in 99.996% D 2 0.

In the study of AK*substrate interactions, nucleotides (with or without metal ions), were mixed with proper amounts of enzyme.

Titration experiments were accomplished by successive addition of components. 30

3.6 Synthesis of Nucleotides

The 170 labeled adenine nucleotides are listed as follows:

0 0 0 0 0 I i 0-P-OAd 0-P-O-P-OAd 0-P-0-P-0Ad i ■ i t i 0 0 0 0 0

[ 1 7 03]AMP [ r - 1 7 o3]adp [a- 1 7 02]ADP

0 0 0 0 0 0 0 0 p l i I 0-P-O-P-O-P-OAd 0-P-0-P-0-P-0Ad 0-P-0-P-0-P-0Ad I I I i I « 1 I i 0 0 0 0 0 0 0 0 0

Cy-1 7 o3]atp O 1 7 0 2]atp [a- 1 7 02]ATP

Schemes 3.1 and 3.2 illustrate the chemical and biochemical synthetic pathway for *7 0 -labeled adenine nucleotides.

isocyanate pl 7 0/* ATP( trace) . | 1. Pl7OCl3 S Myokinas^ADP^ Cnrhamyl-P1703 2. H2 0 AHP(excessT^ y/C YCarbamate — Adenosine - ta- 1703JAHr [ y-1703 jatp coCO2j .+ + mi3 1. (^o)2roci Myoklnas ltrace)

2. ro2‘ 'ADP( trace)

[ a-l;02 ]ADP [ R-17 o, ]ADI [ w-17og ]Arr j 1. 0 2. ro^'

Scheme 3.1’ Scheme 3.2

t 1 7 0 3]AMP This synthesis was accomplished by coupling adenosine 31

with P0C13 (170 labeled trlchlorophosphate) followed by a H20

hydrolysis (Murray & Atkison, 1968; Sammons, 1982).

The crude product after DEAE Sephadex A-25 column, was found to

have two components (Rf: 0.4, 0.6). As shown in Figure 3.1A, the

by-product (Rf: 0.6) at -10.9 ppm was identified as AppA. This was

substantiated by adding AppA, from Sigma, to the mixture, observing

that the peak intensity had increased. The two components could be

separated by silicic acid (Woelm, 32-63 urn) column chromatography

with a mobile phase containing n-BuOHiN^OHr^O (6:3:1, v/v). The

pure [ 1 7 03] AMP is shown in Figure 3 .IB.

A

U

-10 ppm

Figure 3.1 ^PNMR spectra (81.0 MHz) of (A) the mixture of [* 7 03]AMP

and AppA (50 mM, pH 8.0), (B) [ 1 7 03]AMP (50 mM, pH 8.0). Spectral parameters: spectral width 2,500 Hz, acquisition time 1.6 sec, 60°C pulse, line broadening 2 Hz. The broad peaks are due to *70 quadrupolar effect. 32

[a -^ 0 2]ADP The title compound was synthesized by coupling

ancj phosphate. The procedure was implemented according to the reports of Eckstein & Goody (1976) as well as Sammons (1982).

Figure 3.2 shows the ^ P NMR spectra of [ct-^0 2 ]ADP. The ^ 0 - quenched PQ appears at -9.5 ppm, whereas the unlabeled P0 is at -4.8 ppm. The atom % ^ 0 enrichment was estimated as 40 + 356, according to the method of Tsai (1980).

-4 -5 -8 -1 - I -9 -10 P P m

Figure 3.2 ^ P NMR spectrum of [a-* 7 02 ]ADP. The spectral parameters and sample conditions are the same as in Figure 3.1. 33

[B-1 7 03]ADP [B-1 7 0 3]ADP was synthesized biochemically according to published procedures (Mokrasch et a l., 1960; Cohn & Hu, 1980;

Huang & Tsai, 1982). The overall reaction is shown as follows:

AK AMP + ATP (trace) 2ADP

co2 + nh 3 0 / II 1 7 CK / AK ADP + H2 N-C-0-P1 7 03 ± = 4 [y- 1 /03]ATP =*==? [B- 03]ADP + ADP

The carbamyl phosphate was obtained by incubating H 3 P1 7 0 4 (1.2 ml) with 2 portions of KOCN ( 8 ml 1M, 2.4 ml 1M) in 2 ml of KOAc (pH

4.9). This solution was then mixed with a 50 ml, pH 7.3, 0.5M Tris-

C1 buffer solution containing 50 mM AMP (Sigma), 50 mM MgSO^, 0.7 mM of ATP and 25 mM KC1. The reaction was initiated by introducing 700 units of carbamate kinose (Sigma) and 1,000 units of adenylate kinase

(Sigma). The 31P NMR spectrum is illustrated in Figure 3.3. The 170 enrichment was estimated as 44 + 356.

X

0 5 - 1 0 -15 ppm Figure 3.3 3lP NMR'spectrum of [b- 1 7 03 ]ADP. The spectral parameters and sample conditions are the same as in Figure 3.1 34

[o- 1 7 0 2]ATP The preparation of the title compound was achieved by coupling phosphoenolpyruvate and [a-*702]ADP, catalyzed by pyruvate kinase, according to the equation below:

0 0 PK 0 0 0 I I i l l phosphoenol pyruvate + O-P-O-^-OAd =*=? pyruvate + 0-P-0-P-0-l|-0Ad 0 0 0 0 0

0.3 mmole of [a- 1 7 02]ADP was added into 160 ml of 0.1 M Hepes buffer containing 50 mM KC1, 25 mM MgClg and 1 mM DTE at pH 7.5.

Reaction was initiated by introducing 0.4 ml of pyruvate kinase stock solution (ca. 2000 units) and completed after 20 hours stirring. TLC showed only one spot corresponding to ATP. The *70 enrichment was estimated as 38 + 2%.

I | |------1------T------1------»------1------0 - 1 0 - 2 0 ppm

Figure 3.4 31P NMR spectrum of [a-1702]ATP. The spectral parameters and sample conditions are the same as in Figure 3.1. 35

[B-1 7 02]ATP [b-1 7 02]ATP was synthesized according to the procedure of Wehrli (1965) and Lowe (1980).

1.2 mmole DCC (ca. 0.38 g) in 14 ml t-butanol was added dropwise over a period of 3 hours to refluxing aqueous t-butanol ( 6 ml, 1:1 v/v) containing 0.3 mmole of [B-^OglADP and 1.8 mmole (1.8 ml) of morpholine. The mixture was refluxed for an additional 3 hours.

After cooling, N,t'T-dicyclohexylurea salt was precipitated by adding three portions of 15 ml of diethyl ether. The aqueous layer was separated and purified with DEAE-Sephadex A-25 chromatography. ADP morpholidate was eluted as the TEA salt. 0.27 g of 4-morpholine-

N,l'T-dicyclohexylcarboxamidine in dry methanol was then added. The white, gummy solid, bis(4-morpholine-N,fr-dicyclohexylcarboxamidine) salt of ADP-morpholidate, was precipitated by adding 50 ml of dry ether. The residue was rendered anhydrous by adding dry pyridine and rotary evaporated several times. The remaining pyridine was eventually removed by two evaporations with dry benzene.

0.6 ml of n-butylamine was added to 89 ml of pyridine containing

0.115 g of orthophosphoric acid. The clear solution was evaporated to dryness under reduced pressure. The mono(tri-n-butylammonium) phosphate was then rendered anhydrous by evaporating with dry pyridine (3 x 300 ml)followed by dry benzene (2 x 10 ml) and dry tolulene (2 x 10 ml). The phosphate was finally dissolved in rigrously anhydrous DMS0 (2 x 5 ml) and added to the ADP-morpholidate in a dry box. The-clear solution was sealed under dry nitrogen and kept stirring at 35°C for 45 hours. The solution was dilutedwith

0.2 M TEAB buffer and chromatographed on a DEAE A-25 column. The 3*P 36

NMR spectrum of the product is shown in Figure 3.5. Overall yield ~

15%, and 39 atom % * 7 0.

0 10 -20 PP m

Figure 3.5 NMR spectrum of [8 -^ 0 2 ]ATP. The spectral parameters

and sample conditions are the same as in Figure 3.1.

Cy-1 7 o3]atp The title compound was synthesized by coupling ADP

and P^O^ ( * 7 0 labeled phosphate), catalyzed by carbamate kinase

(Tsai, 1980), that is shown by the second eq. on page 36. The

reaction was initiated by adding 1000 units (ca. 40 mg) of carbamate kinase (Sigma). After stirring at 38°C for 4 hours, the ratio of

ADP/ATP was found as 1:1. The ^ P NMR spectrum of Cy-* 7 0 3]ATP is shown in Figure 3.6. The *70 enrichment was estimated as 42 + 2%. 37

10 I dT ppm

Figure 3.6 3*P NMR spectrum of [y-^OgJATP. The spectral parameters

and sample conditions are the same as in Figure 3.1.

3.7 NMR Methods

1*0 NMR Instrumentation

The 170 NMR spectra were obtained at 40.68 MHz on a GE widebore

300 spectrometer. Due to the acoustic ringing (Klntzinger, 1983), a

delay is necessary to apply between the end of the pulse and the

beginning of the acquisition. 30 us of this delay was found to

maximize the FID while minimizing the base line distortion (Rodger et

a l., 1978; Schwartz et a l., 1983). The spectral width was, in

general, 20,000 Hz (ca. 500 ppm), which required 102.4 ms as the acquisition time.

Most experiments were performed with decoupling power off and in

a horizontal non-spinning probe ( 1 0 mm outer diameter, 2 ml sample 38

size). However, the decoupled *70 NMR experiemnts (p. 40) were

done with a spinning horizontal probe (HO mm outer diameter, 4.5 ml

sample size), ^ -d ep lete d water (0.00338 atom % * 7 0, Yeda) was used

as solvent. Tj inversion recovery pulse sequence was employed for

partial suppression of the solvent signals (20.4 mM H2170 of aqueous

solution). The 180°, 90° pulse and t were 52 us, 26 us, and 5 ms,

respectively. A 20 ms delay time was used between acquisitions.

Chemical shifts were referenced to the water signal with a

positive sign indicating downfield shift. The water resonance,

however, is a poor reference because its line width and absolute

frequency are sensitive to temperature (Florin.& Alei, 1967), pH

(Meiboom, 1961), ionic strength (Hertz & Klute, 1970), and

instrumental conditions. It was, therefore, Important to keep

constant sample conditions as well as instrumentation. Probe

temperature control was accomplished by flowing cold air (through dry

ice/i-PrOH bath), maintained at 27°C by a temperature controller. An

artificial line broadening of 50 Hz was applied to process all the

data.

170 NMR Linewidth Measurement

The "corrected" line widths were obtained by subtracting the

contribution of artificial exponential multiplication (50 Hz), field

inhomogeneity (20~30 Hz for horizontal, non-spinning samples)and

other factors such-as spin-spin coupling from the "observed" signal widths. The "observed" signal width was an average from the direct measurement at half-high and the computer calculation. There are two types of spin-spin coupling involved in 170 NMR,

170-1H and 1 7 0-^P. The samples were always maintained at pH 8.0, all the phosphates of ADP and ATP are deprotonated. Therefore, all the 170 NMR experiments were obtained without *H decoupling to avoid rasing the probe temperature. The ^7 0-^^P spin-spin coupling constant, dp_0, was found in the order of 105-120 Hz for ATP measured at elevated temperatures (Gerlt et. el, 1982). It seems that either the observed AO should be corrected or the experiments have to be performed with 31p decoupling to avoid the extra linewidth attributed by l 7 0 - 3lp coupling.

01 Figure 3.7 shows the temperature and ^AP decoupling effect on the 170 NMR signal of [y- 1 7 03 ]ATP. At 55°C, the undecoupled signal qi is resolved, with Jp_o=105 Hz, which collapsed upon J1P decoupling.

AT 30°C, both the undecoupled and decoupled signals are broadened, which is caused by the temperature dropping (see eq. 2.2 and 2.3).

However, the ^p-decoupled one is only 30 Hz sharper than the undecoupled one. Such narowings were found to be mainly caused by a

5-10°C inrease in the actual sample temperature caused by the decoupler power. Thus, the P-0 coupling contributes significantly to the *70 linewidth only when the coupling is partially resolved. When the signal is broad, it can contribute no more than 10-20 Hz, which is within the experimental error.

Based on the above discussion, both the effect of * 7 0-*H or 1 7 0-

3!p coupling on the borad 170 NMR signal of metal ion«nucleotide complexes can be neglected. 40

undecoupled decoupled

150 100 i— i—|— i—i—i—|—i—i—i—(—r —i— i—(—i—i—i—(—i—i—i—|—r 6000 4000 2000 »z 6000 4000 2000 Hz

Figure 3.7 The comparison of undecoupled and 3 *P-decoupled *70 NMR spectra of 20 ymole of [y- 1 7 03]ATP 1n 2.0 ml of 1 7 0-depleted HgO at

(a) 55°C, (b) 30°C. 41

31P NMR

Three different NMR spectrometers were used to obtain

31P NMR data: a Bruker WP 200 (80.1 MHz 3 1 P), a GE widebore 300

(121.5 MHz 3 1 P) and a Bruker AM 500 (202.4 MHz 3 1 P). Chemical shifts were referenced to an external 85% H 3 PO4 standard sample. Samples were always 2.0 ml and contained in a 10 mm NMR tube (Wilmad). D20 was necessary for field frequency lock.

To obtain the very best possible 3*P NMR spectra, the follwing precautions were taken. First, to prevent line broadening due to paramagnetic impurities on the glass wall of the tube, NMR tubes were first soaked in an acid bath for several hours, washed with distilled water and soaked in distilled water for at least 6 hours prior to use. Second, the minimum volume for a compromise between resolution and the economy of samples was just above the receiver coils. Third, 7 3 fine tuning on the probe and shimming on z, z , z coils were extremely important. The sensitivity and resolution were always checked with a standard spectrum of TMP. The best possible instrumental conditions are shown in Figure 3.8. S/N higher than 100 and line width smaller than 0.5 Hz (with LB = 0.2 Hz) was required.

In addition, since the NMR shims were affected by temperature and ionic solution, they were commonly tuned with a DgO sample at the desired temperature. In the study of enzyme^substrate interactions, long time periods for accumulation were usually necessary.

Maintaining the tunning and shiming was very critical to obtain good spectra. Temperature control was also very important in obtaining constant NMR parameters. The best power level that allows for 42

.5 Hr

y w - *tvy\ w \ f i V/

Figure 3.8 NMR- spectrum of the standard TMP (10% in CgDg).

Spectral parameters: spectal width 500 Hz, 1 transition, 90° pulse,

Unebroadening 0.2 Hz, *H decoupling off. 43

decoupling, but does not heat the sample, was 10 H (2 Watts) on the

300 MHz and 12 H (1.5 Watts) on the 200 Hz. LiquidN 2 was employed

to maintain a 10°C probe in the study of AK.

3.8 Calculation of Stoichiometry (Nonlinear Approximation)

Taking a pH=8.0 solution containing ATP and varient ligand L

(i.e. M(11), M(III) and AK), so that the ATP-proton reactions can be

neglected, the major process that take place is the formation of

L(ATP)n complex. The equilibrium and the formation constant involved may be expressed as:

[L(ATP)n] nATP + L ^ L(ATP)n K=------^ (eq. 3.1) [L][ATP] where [L] and [ATP] denote free concentrations and n is the

stoichiometry. The formation constants K, could always be obtained from literature. Thus, given initial concentration of ATP as AQ and the molar ratio of L/ATP as R, eq. 3.1 becomes

Cx] (eq. 3.2)

A computer program solves the n order equation and gives the concentrations of free ATP and L(ATP)n complex. In theory, the observed value (i.e. a NMR parameter) 1s an averaging contribution from those of free ATP and L(ATP)n depending on the relative ratio.

Thus, by knowing [ATP] and [L(ATP)n], the calculated value could be obtained according to eq. 3.3. 44

[ATP] n[L(ATP) ]

Pcacl * Po -KT + P 1 A“ (eq- 3>3» where PQ and Pj represent the observed NMR parameters for free ATP and L(ATP)n. The program then changes in a systematic way the values of the n and repeats the above process until a best f it (in a nonlinear least-squares sense) between the calculated and experimental data is obtained.

In the display of experimental results, the calculated titration curves together with the data points are plotted vs. R (the ratio of tL]/[ATP]). CHAPTER IV

THE INTERACTION OF M (II) WITH ADP AND ATP

4.1 3*P NMR Properties of Alkaline Earth M(II)ADP and H(II)ATP

In a pH 8.0 solution which contains M(11) and nucleotide L (ADP or ATP), all the phosphate groups will be deprotonated, and the

equilibrium can be represented in simplified form as:

nL + M «a= 5s M(L)n

where n is the stoichiometry. In this section, the titration curves of 3*P NMR chemical shifts of ADP and ATP as a function of

[M(II)]/[ADP] or [M(II)]/[ATP] are used to determine the stoichiometry, n, shown in the equilibrium above.

Figure 4.1 shows the 3*P NMR spectra of ADP free and titrated with varying concentrations of Ca(II). The corresponding chemical shifts and coupling constants are listed in Table 4.1, together with the parameters for other M(II)ADP and M(II)ATP complexes. A single set of resonances representing the average of free and metal bound

ADP indicates that the exchange rate is fast on the 3lP NMR time

45 46

[Ca(11) ] /[ ADP]

0.2

0.5

U- 0.8

1 .0

1 .2

1 .5

2 -4 -6 -B 10 12 -14 PPM

Figure 4.1 3*P NMR spectra of ADP with (10 mM, pH 8.0)varying concentrations of Ca(II). Spectral parameters: spectral frequency

121.47 MHz, spectral width 1500 Hz, acquisition time 1.36 sec, 8 K data points, acquisition delay 1.0 sec, 30° pulse angle, line broadening 1.0 Hz, ambient temperature (22±2°C). 47

Table 4.1 3*P NMR Parameters of M(II)ADP, M(II)ATP Complexes

at pH 8.0

Chemical Shift Change (ppm)a Coupling Constant

Complexes p« PB PY JctB J f*Y 1

ADP (- 1 0 . 1 ) (-5.7) 22.7 Mg(II)ADPb +0.5 +0.14 17.5

Ca(II)ADPc +0 . 8 +0.7 19.8

Sr(II)ADPc +1 . 0 +1 . 1 19.8

Ba(II)ADPC +1 . 1 +0.9 2 0 . 6

Zn(II)ADPd +1 . 1 +0.4 16.8

Cd(II)ADPe +1 . 6 +1.9 19.1

Hg(II)ADP + 0 . 1 +0 . 1 22.5

ATP (-10.4) (- 2 1 . 1 ) (-5.5) 19.6 2 0 . 0 Mg(II)ATPf +0.3 +2.5 +0.5 15.8 15.8

Ca(II)ATP +0 . 2 +2 . 0 +0 . 6 16.6 17.2

Sr(11)ATP +0.4 +1.9 +1 . 2 17.2 17.4

Ba(II)ATP +0 . 2 +0.7 +0.5 18.9 19.1

Zn(11)ATP +0.4 +2 . 2 +0 . 8 16.4 16.0 Cd(II)ATP +0.4 +2.4 +1.5 17.7 15.7

Hg(II)ATP +0 . 1 +0 . 1 0 19.3 19.2 a"+" sign indicates downfield shift from the signal of free nucleotides. bData obtained from Tran-Dinh and Neumann (1977). cFrom titration curves in Figure 4.2,A,B,C. dFrom titration curves in Figure 4.10. eFrom titration curves in Figure 4.8.

^Data obtained from Bock (1980). 48

scale. Both Pa and P 6 peaks shift downfield relative to their metal-

free state. Taqui Kahn & Martel 1 (196E) has reported the stability constants for various M(11) -ADP complexes (see Table 4.3). Based on p the reported stability constant (7.3 x 10 at pH 8.0), a nonlinear

least square fitting program was applied to fit the data by entering an initial value of 1.0. n values of 0.99 and 0.99 were thus obtained for the fitting of PQ and Pg, respectively. The statistical

results are shown in the Table 4.2 and the appendix at the end of text. The plot of the theoretical and experimental chemical shifts vs. the ratio of [Ca(II)]/[ADP] is shown in Figure 4.2,A. The plateau beyond [Ca(II)]/[ADP] = 1.0 supports a 1:1 stoichiometry of

[Ca(11)]:[ADP]. Thus, the formation of Ca(11) -ADP at a 10 mM concentration range, and pH 8.0, is believable.

Figure 4.2,B,C show similar titration experiments of ADP with

Sr(II) and Ba(II). In all cases, both PQ and Pg signals shift downfield. As shown in Table 4.1, almost all divalent metal ions cause the downfield shift, however, the mechanism is unclear. The

fitting results (Table 4.2) also show 1:1 stoichiometry.

Figure 4.3,A shows the 3*P NMR spectrum of 10 mM ATP, pH 8.0.

The introduction of varying amounts of Sr(II) (spectra B~H) causes downfield shifting of Pa, Pg, and P^ resonances. Based on the 3 1 assumed stability constant (3.46 x 10 M " 1 Taqui Khan & Martell,

1966), n values (stoichiometry) of 1.00, 1.02, and 1.05 were obtained for the fitting of PQ, Pg and P^, respectively. The plot of chemical shift changes vs. [Sr(II)]/[ADP] is shown in Figure 4.4. Table 4.2 Nonlinear Least Square Fitting Results for Stoichiometry

of M(11)«ADPa and M(II)ATPb

ADP ATP

Pa Pa PR PB PY

Ca(II) 0.99 + 0.05 0.99 + 0.05 0.99 + 0.02 1.03 + 0.04 1.12 + 0.03

Sr(II) 1.09 + 0.02 1.00 + 0.05 1.00 + 0.04 1.02 + 0.05 1.05 + 0.04

Ba(II) 1.14 ± 0.02 1.11 i 0.02 1.21 ± 0.05 1.17 ± 0.06 1.25 + 0.04

Zn(II) 1.09 ± 0.11 1.05 ± 0.02

Cd(II) 1.06 ± 0.08 1.04 ± 0.05

a The fitting was based on stability constant of 7.3 x 10 2 M”* for

Ca(II)ADP, 3.5 x 102 M_1 for Sr(II)ADP, 2.3 x 10 2 M" 1 for Ba(Il)ADP,

1.9 x 104 M" 1 for Zn(II)ADP (Taqui Kahn & Martel 1, 1962), and 3.8 x

103 M"* for Cd(11)ADP (Pecoraro et al. 1984).

b 9.3 x 103 M- 1 for Ca(11)ATP, 3.5 x 10 3 M' 1 for Sr(II)ATP, 2.0 x

103 M" 1 for Ba(II)ATP 7.0 x 10 4 M- 1 for Zn(II)ATP (Taqui Kahn &

Martell, 1966), and 2.3 x 10 4 M" 1 for Cd(II)ATP (Pecoraro et al.

1984). 50

11.0

10.0

9.0

0.4 0.B

f Ca(II) J/[ADPJ

Figure 4.2.A Titration curves of 31P NMR chemical shifts of ADP (10 mM, pH 8.0) with CaC^* 1 1 .0

ppm

6.0

5.0

0.4 0.8

[Sr(II)]/|ADP]

Figure 4.2B Titration curves of 31P NMR chemical shifts of ADP

(lOmM, pH 8.0) with SrC^. 1 1.0

9.0

PPM

6.0

5.0

0.4 0.8 1.2 1.6

I Ba(11) ] / { ADP]

Figure 4.2.C Titration curves of ^*P NMR chemical shifts of ADP

(lOmM, pH 8 . 0 ) with BaCl2- 53

[Sr(II)]/[ATP J

P,

0.2

0.6

i^W AAV>y*M< 0.8

UA/*yt*»»> 1 * 2

1 1.3

1 .5

t — i— i— r i—i—r I 1 1 1 ' I T t— i— I— | — i— r -5 10 -15 -20 -25 PPM

Figure 4.3 ^lp NMR spectra of ATP with varying concentrations of

Sr(II). Sample condition and spectral parameters are the same as In

Figure 4.1. 54

.0

19.0

9.0

6.0

4.0 0.4 0 .B 1 .2 1 .6

[Sr C11) J/ L ATPJ

Figure 4.4 Titration curves of 31p NMR chemical shifts of ATP with

SrCl2. 55

In the titration experiments of ATP with Ca(II) and Ba(II), the

fitting results and the plateau of the 3*P titration curves after

[M(II)]/[ATP] = 1.0, again, suggests 1:1 stoichiometry for these

M( 11)ATP species.

In summary, the 1:1 complexes of M(11) -ADP and M(11) -ATP is

established. This stoichiometry of alkaline earth metal ions

complexed with ADP and ATP had been proposed by potentiometric

titration (Taqui Khan & Martel 1, 1962, 1966). The consistency of our

results suggests the accuracy of the 31P NMR chemical shift method in detecting the stoichiometry of metal ion*nucleotide complexes.

However, as pointed out in Section 1.1, if one attempts to

interpretate the structures of those M(II)»nucleotide complexes by

3*P NMR chemical shift changes, the results can be questionable. For example, in the case of MgATP and CaATP, A 6 Pa< A6 PY<< A6 P0 is observed. Again, this does not mean that these metal ions coordinate predominatly to the B, phosphate of ATP. As an alternative method,

170 NMR will be employed in Section 4.2 to address these problems.

4.2 170 NMR Properties of Alkaline Earth M(II)ADP and M(II)ATP

Prior to the discussion, I would like to point out that the data quality is limited by the experimental error which was in turn caused by the inherent broad signals. However, if the data is only for qualitative or semiquantitative analysis, the decription is adequate and the result is acceptable. Figure 4.5 shows the *70 NMR spectra (40.68 MHz) of [a -^ 0 2 ]ADP,

[B -^ 0 3 ]ADP and their corresponding M(II) complexes with the ratio of

[M(II)]/[ADP] = 1.05. Since 1:1 stoichiometry was observed, 90% of the nucleotides are present as metal bound form. The NMR parameters for these complexes are listed in Table 4.3. Comparison of the *70

NMR properties of the M(11)ADP complexes was made at pH 8.0, at which the free and complexed ADP were in the fully ionized form. Ca(II),

Sr(II), and Ba(II) induce upfield shifts, however, Mg(11)ADP is the only complex which shifts downfield. The reason for this chemical shift behavior is unclear. For each complex, binding of M(II) causes the increase of the *70 NMR linewidths (See Section 2.3 for a rationale for the increase of linewidths).

The "170 NMR line broadening effect" induced by the coordination of metal ions has been defined (Huang & Tsai, 1982., Sammons et al.,

1983) as:

where AOf and A^ represent the "corrected" 170 NMR line widths of free and bound ATP. As shown in Table 4.3, it is obvious that the R values-of a-*70 and R-*70 for M(II)ADP follow the order: Mg(11)ADP >

Ca(11)ADP > Sr(11)ADP > Ba(II)ADP. Since 8-*70 has a smaller AOf than a-1 7 0, the fact that the R values of R-170 are larger than those of a - 1 7 0 for all complexes, does not necessarily suggest that

M(11) interacts with the R-phosphate to a greater extent than thea - phosphate. The present 170 NMR data suggest that not only Mg(II)ADP, but also Ca(II)ADP, Sr(II)ADP, and Ba(II)ADP are predominantly a , B- 57

FREE FREE

T T TT T T T T TT 200 100 200 100

CHEMICAL SHIFT (ppm)

Figure 4.5 170 NMR spectra (40.68 MHz) of [a- 1 7 02]ADP and [B-

* 70 3 ]ADP, and the corresponding complexes with alkaline earth metal ions. Sample condition: 10 mM ADP, 10.5 mM MUljClg. pH 8.0, in

* 7 0-depleted water (2 ml). Spectral papameters: spectral width

20,000 Hz, acquisition time 102.4 msec, receiver gate 30 usee, line broadening 50 Hz, 4 K data points. The Tj inversion-recovery experiment was used for partial suppression of the HgO signal (180°, t, 90° = 52 ysec, 5 msec, 26 gsec, respectively). The delay between acquisition was 20 msec. The temperature was regulated at 2U°C. 58

bidentate, although a small percentage of 0 -monodentate complexes

cannot be totally ruled out.

The 170 NMR experiments of Mg(II)ATP, Ca(II)ATP, Sr(11)ATP and

Ba(II)ATP have also been carried out, and the results are also listed

in Table 4.3. Figure 4.6 shows the effect of Mg(II), Ca(II), Sr(II),

and Ba(II) on *70 NMR of [a-^t^jATP, [S-^OgiJATP, and [y-^O-^ATP.

In all cases, linewidth increase caused by M(11) interaction are

observed. The line broadening effect for the a-phosphate of ATP is

smaller than those of 8 - and y-phosphates. Therefore, these results

1n Table 4.3 are reasonable interpreted as the existence of a mixture

of o, 8 , y-tridentate and 8 ,y-bidentate. The ratio of the two species

should also depend on the ionic radii (i.e. Sr(II), Ba(II) have a

high percentage of a* 8 , y-tridentate). It is obvious that the line

broadening effect for M(11)ATP follow the order Mg(II) > Ca(II) >

Sr(II) > Ba(II) (see Table 4.3).

In all M(11)ADP and M(11)ATP complexes, only one *70 NMR signal

has been observed. This signal is most likely due to an average of

1 7 0=P-0" M( 11) and 0=P-* 7 0“ M(II). I will discuss this in detail

1n Section 5.4.2. The line-broadening effect has been observed in

complexes on the slow-exchange limit (CoATP), on the intermediate

range (MgATP), and on the rapid-exchange limit (CaATP) (Huang & Tsai,

1982). Thus, the observed 11ne-broadening effect should also not due to chemical exchange processes (Huang & Tsai, 1982). 59

Table 4.3 Summary of *70 NMR Results

aof or A0h ( Hz )a R Values 660 (ppm) b

Complex a B Y a B Y a B Y

ADP 510 340 —— (93.8) (106.5)

Mg ( 11) ADP 860 $50 0.69 0.91 -4.2 -4.8

Ca(11)ADP 730 525 0.43 0.54 +1.2 +1.8

Sr( 11) ADP 565 430 0.11 0.26 +0.5 +1.0

Ba(II)ADP 570 405 0.12 0.19 +1.3 +3.6

Zn(11)AOP 1075 1775 1.11 4.22 -1.5 -7.0

Cd(I I) (ADP)2 725 1150 0.42 2.38 0 -4.5

Cd(II)2(ADP)2 1180 1650 1.31 3.85 -1.6 0

H g (1 1) AD P 590 410 0.15 0.20 -1.5 +0.8

ATP 480 430 290 — -- (93.3) (100.3) (105.5)

Mg(lI)ATP 780 840 700 0.63 0.95 1.41 -5.7 -8 .2 -4 .5

Ca(11)ATP 690 710 600 0.44 0.65 1.07 +1.5 +2.6 +2.3

Sr(Il)ATP 680 680 460 0.42 0.58 0.59 +1.7 +4.0 +2.5

Ba(11)ATP 590 620 400 0.23 0.29 0.38 +0.8 +5.7 +4.7

aThe line widths were obtained from Lorentzlan line fitting and corrected for artificial line broadening (50 Hz) and field Inhomogeneity (25 Hz). The estimated error Is t 5X. bThe numbers In parenthesis are absolute 60 for free ADP, ATP. "+" and signs indicate downfield and upfleld shifts, respectively, from the corresponding signals of free ADP,

ATP. The estimated error Is t 0.5 ppm. 60

[ a - 1702]ATP [B -1702]ATP [ y - 1703]ATP

FREE FREE FREE

100 100 100 ( ppm)

Figure 4.6 170 NMR spectra ( 4 0 .6 8 MHz) of [a-1702]ATP, [b-1702]ATP, and [y-*703]ATP, arid the corresponding complexes with alkaline earth metal 1ons. Sample condition and spectral parameters are the same as in Figure 4 . 5 . 61

4.3 The Comparison of * 70 NMR R Values and S ta b ility Constants

Table 4.4 shows the log K values for M(II)ADP and M(11)ATP (K: stability constants) obtained by three different methods. In the complexes of ATP and ADP with alkaline earth metal ions, it is obvious that the log K for M(II)ATP are greater than those for

M(11)ADP by a factor of ca. 10 in any one set of the data. The order of K is rationalized by the following two reasons: (i) the

Table 4.4 Logarithms of the Stability Constants {log K) of M(11)ADP and M( 1 1 )ATP

Method Potentiometric NMR

Titration 3 Chemical Shifts^ ^*P NMRC Nucleotide ADP ATP ADP ATP ADP ATP

Metal Ion:

Mg(II) 3.17 4.22 3.20 4.27 4.11 4.70

Ca(II) 2 . 8 6 3.97

Sr(II) 2.54 3.54

Ba(II) 2.36 3.29

Zn(II) 4.28 4.85

Cd(II) 3.58 4.36

aTaqui Khan & Martel 1, 1962, 1966.

^Scheller et a l., 1981; Scheller & Si gel 1983. cPecoraro et a l., 1984. 62 association constants of the alkaline earth metal ions complexed with oxygen-containing ligands is expected to follow the order Mg(Il) >

Ca(II) > Sr(II) > Ba(II) (Huheey, 1978); and (ii) the net charge"-4" and "-3" for ATP and ADP, at pH 8.0, differentiates the binding stability of M(II)ADP and M(II)ATP. The difference of these K values reflects that the interaction between alkaline earth metal ions and nucleotides could be mainly due to the electrostatic interactions between the alkaline earth metal ions and the phosphate moiety of adenine nucleotides. The line broadening effect which is represented by R values also follows the order Mg(II) > Ca(II) > Sr(II) > Ba(II), and (see R values in Table 4.3) seems to parallel the stability constants. Thus, the line broadening effect is a useful parameter in comparing the affinity of different metal ions for nucleotides.

4.4 The Interaction of Cd(II) with ADP

On the basis of NMR chemical shifts, Bock (1980) has suggested the following equilibrium:

Cd(II) Cd(II) 2ADP Cd(II)(ADP )2 - Cd(II) 2 (ADP) 2

In light of the study, I repreated the 1H NMR titration experiment of ADP with Cd(II) (Section 4.4.1), ^*P NMR was used to support the stoichiometry concluded from *H NMR results (4.4.2), and

170 NMR was employed to probe the sites of coordination in

Cd(II)(ADP) 2 and Cd(11) 2 (ADP) 2 complexes (4.4.3). 63

4.4.1 lH NMR

In Bock's report, the curves were determined with only five titrations, and the ratio of [Cd(II)]/[ADP] was only carried out up to 1.0. The experiment was repeated with more data points in the range of [Cd(II)]/[ADP] = 0 ~ 1.5. Figure 4.7 shows the effect of

Cd(II) on the lH chemical shifts of ADP. Three stages corresponding to the ratio of CCd(II)]/[ADP] = 0 ~ 0.5, 0.5 ~ 1.0, and 1.0 ~ 1.5 are observed. In the first series of additions of Cd(II) (i.e.

[Cd(II)]/[ADP] = 0 ~ 0.5), Hg is shifted downfield, suggesting the Ny coordination (Cohn & Hughes, 1962., Scheller & Sigel, 1983., Scheller et al., 1981), and H£ is shifted upfield, most likely due to a ring current effect caused by "base stacking", however, Cd(II) has little effect on ribose protons. Upon addition of the second series of

Cd(II) aliquots (i.e. [Cd(II)]/[ADP] = 0.5 ~ 1.0), Hg and H ^ remain unchanged, whereas all the ribose protons are shifted upfield. The above observation is consistent with the literature (Bock, 1980). In addition, our study shows that further addition of Cd(II) (i.e.

[Cd(II)]/[ADP]>1.0) does not perturb the chemical shifts of any protons.

The chemical shift behavior of Figure 4.7 can be interpreted as follows: the first half-equivalent of Cd(II) induces the formation of a Cd( 1 1 )(ADP) 2 complex, in which there is a direct interaction between the Cd(II) ion and the Ny on both rings. A second Cd(II) may then bind to this complex to form a Cd( 1 1 )2 (ADP) 2 species. The plateau of titration curves beyond [Cd(II)]/[ADP] = 1 .0 illustrate that the formation of higher order Cd*ADP complexes is less probable. 64

6 ppm

Cd(ll) 'ADP

Figure 4.7 Titration curves of *H chemical shifts of ADP (10 mM in

D2 0, pH 8.0) with Cd(II). Spectral parameters: spectral frequency

200 MHz, spectral width 2000 Hz, acquisition time 4.1 sec, 16 K data points, line broadening 0.4 Hz, ambient temperature (30 ± 2°C). 4.4.2 3lP NMR

Figure 4.8 shows the 3*P NMR chemical shifts of ADP as a function of the ratio of [Cd(II)]/[ADP]. Both Pa and PR signals are shifted downfield and reach plateau after [Cd(II)]/[ADP] =1.0. The nonlinear least square fitting program suggested a 1 : 1 stoichiometry

(Table 4.2). However, this stoichiometry cannot exclude the existence of Cd»ADP or any higher order Cd(I I )n(ADP)n complex. The O 1 failure to observe Cd(ADP ) 2 is probably because the P chemical shift changes for different complexes are not significant enough to be differentiated from experimental error. This demonstrates again the ineffectiveness of the 3*P NMR chemical shift method in the study for the structure of metal ion«nucleotide complexes.

Instead, the *70 NMR line broadening effect is probably a good substitute method to address this problem.

4.4.3 l70 NMR

The 170 NMR experiments of [a-l 7 02] and [b- 1 7 03]ADP titrated with Cd(II) are shown in Figure 4.9, a and b. The linewidths AO, and chemical shifts 60, are plotted as a function of [Cd(II)]/[ADP], In the range of [Cd(II)]/[ADP] = 0 ~ 0.5, the chemical shifts of a -1 7 0,

(60, 93.8 ppm) remains unchanged, the linewidth increases simply from

510 to 725 Hz. Meanwhile, 60 of B-*70 shifts downfield from 106.5 to

1 0 2 ppm, A0 increase from 3 4 0 to 1 1 5 0 Hz. It is obvious that the a 0 17 17 and 60 of a - '0 are.less affected than B- 0 in this range (i.e. the addition of the first eq. of Cd(II)). Such results support the 66

10.0

0

.0

.0

0.4 0.8 1 .2 1 .6

| CdC1 1 ) 1 / IADP]

Figure 4.8 Titration curves of 31P chemical shifts of ADP (10 mM, pH

8.0) with Cd(11)2. Spectral parameters are the same as Figure 4.1. 67

So 106 * (ppm) |0 4 _

1 02 A 95 -1 I /?-,7o

i /3 -l7o

1500-

10001

500;

0.5 1.0 Cd (II)/ADP

Figure 4.9 Titration curves of 170 NMR line widths (a) and chemical shifts (b) of [a-l^DADP and [b-* 70 3 ]ADP with Cd(II). Spectral parameters are the same as Figure 4.5. formation of the first complex, Cd(11) (ADP)2* as suggested by

NMR. In addition, the 170 line broadening effect of p-170 indicates that Cd(II) coordinates with P 0 of both ADP predominantly. Upon addition of the second half-equivalent of Cd(II), the R values for a and p-170 signal increase from 0.42, 2.38 to 1.31 and 3.85 respectively. This suggests the coordination of Cd(II) to both the a- and p-phosphate of ADP. Thus, the second complex Cd(II) 2 (ADP) 2 i formed.

Since the stability constant of Cd(11)ADP is smaller than that of Mg(II)ADP (see Table 4.4, Pecoraro et a l., 1984), we expect the R values of Cd(11)ADP to be smaller than those of Mg(11)ADP if the correlation between R values and stability constants observed for alkaline earth metal ions holds. The relatively large R values of

Cd(II)ADP could be due to an increased rotational correlation time,

T r , which in turn resulted from the self-associated complexes of

Cd(11)(ADP ) 2 and Cd(II) 2 (ADP)2. Furthermore, the gradual increase i the 40 of a -^ 0 from 0 to 0.5 equivalents of Cd(II) is likely to be due to an increase in xp instead of a small percentages of a- coordination.

Cd

Cd(RDP)2 Cd2 (RDP)2

Scheme 4.1 69

As shown in Scheme 4.1, in summary, ^ 0 and NMR results suggest that in the Cd(11)(ADP ) 2 complex, Cd(II) binds to both Ny and

of two ADP molecules and the two adenine rings are stacked. In the Cd(II)£(AOP)g complex, it is most likely that one Cd(II) ion binds to Ny and Pg of two ADP, while the second Cd(II) ion binds to both P and PD of two ADP molecules. The conformation of the a r adenosine moiety is somewhat different between the two complexes, so that the ribose ring is exposed to the base in the second, but not in the firs t.

4.5 The Interaction of Zn(II) and Hg(II) with ADP

4.5.1 The NMR Properties of Zn(II)«ADP complex

The titration curves of the 31P chemical shifts of ADP with

Zn(II) are shown in Figure 4.10. The coordination of Zn(II), like all other M(11), causes the downfield shift of the Pa, Pp of ADP.

However, the Pg signal is affected to a smaller extent than the PQ signal. Both lines are slightly curved, which resemble the titration of ADP with Cd(II), and show no clear evidence for different complexes. Nonetheless, the chemical shifts remain unchanged beyond

[Zn(II)]/[ADP] = 1.0 suggest a 1:1 stoichiometry. (See Table 4.2 for fitting results.)

The NMR property (Figure 4.11) is similar to that of

Cd(II)ADP. Except Hg» all resonances shift upfield. However, the chemical shift changes are continuous in the region [Zn(II)]/[ADP] =

0 ~ 1.0, and the curves look smoother. Thus, no 1:2 complex can be clearly identified. 0

.0

0

0

°*4 0.8 1.2 1.6

[Zn(11) ] / IADP]

Figure 4.10 Titration curves of 3lP chemical shifts of ADP with

Zn(II). Spectral parameters are the same as In Figure 4.1. 71

6 ppm

H

i____ 0.2 0.6

Z n (m ^

■— ^ A D P

Figure 4.11 Titration curves of chemical shifts of ADP (10 mM in

D2 0, pH 8.0) with Zn(II). Spectral parameters are the same as in

Figure 4.1. 72

SO . (ppm) 104 _

102

100 -

1500 -

1 0 0 0 -

0 0 . 5 1.0 Zn(U)/ADP

Figure 4.12 Titration curves of *70 NMR line widths (a) and chemical shifts (b) of [a-^OgJADP and [P-^OgDADP with Zn(II). The spectral parameters are the same as in Figure 4.5. 73

Figure 4.12 shows the titration curves of *70 NMR linewidths and

chemical shifts of [a-^^ilADP and [b-^OjJADP with Zn(II). The *70 NMR

titration curves of aO are also somewhat curved. Unlike Cd(II)ADP,

the AO of both a- and R-170 increase contiguously, with the effect on

B- 1 7 0 greater than that on a- 1 7 0. This indicates the involvement of

both P„a and Pa p in the interaction with Zn(II). There is no clear change at [Zn(II)]/[ADP] = 0.5. The slight increase of the a-*70

slope and the slight decrease of the r - 1 7 0 slope between

[Zn(II)]/[ADP] = 0.75 ~ 1.0 qualitatively resembles Cd(II)ADP.

However, this data is not strong enough to identify the different complexes of Zn(II)-ADP.

Since the M(11) ions become "softer" from Zn(II) to Hg(II)

(Huheey, 1978), the tendency to interact with nitrogen ligands at the

adenine ring follows the order: Zn(II) < Cd(II) < Hg(II). The affinity of Zn(II) to Ny is obviously lower than that of Cd(II)

(Scheller et a l., 1981), therefore, it should have a smaller tendency to form dimeric complexes through an intermolecular metal ion bridge.

Taqui Khan & Martel 1 (1962) reported log K (K, stability constant) of 4.28 and 3.17 for Zn(11)ADP and Mg(II)ADP. Whereas

Pecoraro et al. (1984) reported 4.11 and 3.58 for Mg(II)ADP and

Cd(II)ADP, respectively (see Table 4.4). The stability constant of

Zn(11)ADP should be greater than that of Cd(11)ADP. The R values of

Zn(II)ADP are, therefore, expected to be greater than those of

Cd(II)ADP (see Section 4.3). The comparable R values between Zn(II) and Cd(II), however, is an indication that Zn(II)ADP consists of a smaller percentage of dimeric species than Cd(11)ADP. 74

In summary, the smooth curved ^H, 170 and 31P titration curves cannot differentitate several species such as Zn(11)ADP, Zn(II)ADP 2 , and Zn(II) 2 ADP2 . Thus, in the range [Zn(II)]/[ADP] = 0 ~ 1.0, Zn*ADP exist as a mixture of several species.

4.5.2 The Interaction of ADP with Hg(II)

Figure 4.13 shows the titration of *70 labeled ADP with

Hg(II). It seems that both a- and e-170 signals are less perturbed

by the addition of Hg(II). The 3*0 and *7P NMR data of Hg(11)ADP are

listed in Table 4.1 and 4.3, respectively. All the results above

suggest little interaction of Hg(II) with the diphosphate moiety of

ADP. This has been suggested by Bock earlier (1980), and probably

related to the "softness" of Hg(II). As shown in Figure 4.14, the

chemical shifts were also unperturbed, but the H2 and HJ signal was

broadened upon successive additions of the Hg(II) ion. The peak high

ratio of H 2 /H0 remains unchanged after [Hg(II)]/[ADP] = 0.8 suggests that Hg(II) forms 1:1 complex with ADP.

4.6 Discussion

4.6.1 Criticism of NMR Methodology

Compared to the previous NMR study in this field, the progress that we made was the combination of the three different NMR

OI 1 7 techniques. The stoichiometry was determined by P NMR. 0 NMR

results were used to interpret the metal ion binding site. *H NMR, however, was employed to observe the metal ion interaction with the base ring as well as the self-stacking problem. For each NMR method, both advantages and limitations exist in both experiment and 75

[a -1702]ADP [B - 1703]ADP

0,85

0.65

I—I ! - | — i— i— i— »‘ 200 100 100 PPM

Figure 4.13 170 NMR spectra of [a- 1 7 02] and LB- 1 7 0 3 ]ADP titrated with varying concentrations of Hg(II). 76

lHg(II))/[ADPJ

— JV

0.2

0 . 4

0.6

0 .B

1.0

1 .2 JL

_J ______I ■ I------I------1------1------L.

B.O 6.0 4.0 2.0 PPM

Figure 4.14 NMR spectra of ADP (lOmM pD = 8.0), and titrated with varying concentrations of HgClg. 77 interpretation. Fortunately, the advantage of one method usually happens to be the disadvantage of the other two. In this section, I would like to criticize these methods individually.

From the consideration of experiment, ^lp NMR is very practical.

The natural abundance of the nucleus is 100%, and the adenine nucleotides are commercially available. The samples can be prepared in HgO. With 10 mM concentration, any high resolution FT NMR spectrometer will obtain a well resolved spectrum without difficultly.

However, as I already discussed in detail in Section 1.1, the main

0 1 problem lies in the data interpretation. Since AP nuclei do not coordinte to the metal ions directly, the observed parameters may only reflect a conformational change instead of metal ion binding.

Nonetheless, the determination of stoichiometry is still effective by this method.

The major advantage of the *70 NMR methods is that the ^ 0 nuclei are directly involved in metal binding and that the quadrupolar relaxation is sensitive to the close environment of the * 7 0 nuclei.

Thus, the "line broadening effect" caused by the interaction of metal ions is reliable in the interpretation of binding site specificity. Unfortunately, due to the low natureal abundance of the

170 isotope (0.37%), nucleotides have to be synthesized which is tedious and expensive. In addition, a special high power instrument is needed for the observation of quadrupolar nuclei. Therefore, the experiment is somewhat limited by the consideration of economics.

The observation of the weak interactions is the most troublesome.

Sometimes, it is diffiuclt to judge whether the increase of 78 linewidths are due to the metal ion interactions or caused by experimental error.

NMR has both the advantage of sensitivity and accuracy.

However, two aspects have to be considered. Firstly, the repeating lypholization is a necessary process to obtain a solvent (HOD) signal suppressed spectrum. Unfortunately, long time incubation of ATP and

ADP with metal ions usually causes the decomposition of nucleotides.

Secondly, the observation is limited to the base and ribose parts.

The most important moiety, the phosphate chain, cannot be probed by this technique.

4.6.2 The Self-Association of ADP Caused by Cd(II) and Zn(II)

It is well-known that some metal ions such as Zn(II) and Cd(II) promote the self-assocation of adenine nucleoties (Sigel et al.,

1984; 1983; 1982; Scheller & Sigel, 1983). This could occur in the physiological concentrations of some living systems, for example, the chromaffin granules in the adrenal medulla. Different structures of metal ion»nucleotide complexes will be present if the association occurs. As far as the enzyme specificity concerned, this is not acceptable. The self association of nucleotide via intermolecular metal ion bridge has been well studied by *H NMR in the past (see

Section 1.1 for a rationale). In this research, we contribute the knowledge to this intramolecular bridge by 170 NMR technique.

Compared to the previous *H NMR reports, the coordination sites on phosphate chain are the major concern. In Sections 4.4 and 4.5, the

170 NMR results show that Cd(II) and Zn(II) interact differently with 79

PCT and Pp of ADP 1n different complexes.

Scheller & Sigel (1983) proposed the following equilibrium for

Zn(11)ADP and Cd(II)ADP:

M( 11) ADP--* M(II)o(ADP)o ------f M( 11) (ADP) x

M (II)A D P 0 p

The simplified representation of M(11)ADPC^ and M(11) 2 (ADP) 2 are shown in Scheme 4.2. M(II)ADP0p is the "open" monomeric form with

M(II) binding only to the phosphate; M(II)ADPcj is the "closed" monomeric form with M(II) binding to N 7 and the phosphate

Intramolecularly; M( 1 1 )2 (ADP) 2 is the dimeric form with

Intermolecular coordination to N 7 and the phosphate moiety with the adenine rings stacked; M(II)X(ADP)X is the oligomeric form due to further association of the dimeric form.

M(11)ADPc l ' M(II)2 (ADP) 2

Scheme 4.2 80

It was estimated that at infinite dilution, 41% of Cd(II)ADP and

20% of Zn(II)ADP, are in the closed monomeric form, and the percentage of the closed form varies with the series Mn(ADP) <

Co(ADP) < Ni(ADP) < Cu(ADP) < Zn(ADP). This is probably due to the increasing ratio of charge/ionic radius, and in turn reflects the different affinities of these metal ions towards ligands with N donors.

In sections 4.4 and 4.5, we present evidence to support the presence of Cd(II)(ADP ) 2 and Zn(11)(ADP)£• However, these

M(1 1 )(ADP) 2 complexes have been neglected in Scheller & Si gel's report (1983). It seems more reasonable that the equilibrium for

Zn(11)ADP and Cd(11)ADP is represented by:

M(II)ADPcl *=5? M(II)(ADP) 2 *=* M(II) 2 (ADP) 2 =*=5= M(II)X(ADP)X

M(II)ADPop

Based on our experimental results and the discussion above, the formation of Cd(II)(ADP ) 2 and Cd(I I )£(ADP ) 2 complexes are presumably due to the high affinity of Cd(II) for the Ny of the adenine ring.

The lower affinity of Zn(II) to Ny should result in a less tendency to form dimeric complexes such as Zn(II)(ADP ) 2 and Zn(11) 2 (ADP)2 •

It seems that the binding of metal ions increases the self­ association of nucleotides in two ways: some metal ions, like alkaline earth metal ions promote stacking simply by the reduction of charge repulsion as coordinating to the phosphate moiety. Others, 81 like Cd(II) and Zn(II), due to the significant coordination tendency towards both nitrogen and phosphate, enhance the dimers by reducing the repulsion as well as forming an intermolecular bridge between the phosphate residue of one nucleotide and the base ring of the second one.

Apparently, Zn(11)ADP and Cd(II) ADP belong to the latter kind of promotion. Certain other metal ions, like Cu(II), hae stronger coordination tendency toward both the nitrogren on the base ring and the phosphate groups (Schiller et a l., 1981). In those cases, metal ions might be even more effective in the promotion of stacking.

With these discussions in mind, it seems that the extent of base-metal ion interaction varies from metal ion to metal ion and also related to the concentration range (Martin & Mariam, 1979;

Scheller et al., 1981). Thus, the self-association varies from insignificant traces to nearly 1 0 0 %.

4.6.3 Hhy Mg(II)ATP is Chosen for Most Kinases

On the basis of the present results and the reports of others, I attempt to answer the question "why is Mg(11)ATP the natural substrate for most kinases?". We approach this solution from the structures and properties of various metal»nucleotide complexes, and the effect of different metal ions on the catalytic reactions of kinases.

A fundamental rule for metal ions functioning as a cofactor is that the coordination must be strong enough to enforce a conformational 82

change of the phosphate chain. The monovalent, group 1A, metal ions

are not suitable since the charge is not strong enough toform stable complexes with nucleotides. There is, therefore, little chance for

M(I)ATP to approach the transition state. Most divalent transition metal ions, including group 2B ions and Cu(II), Ni(II), Co(II) and

Mn(II), have different degrees of tendency to interact with N 7 . The

self-association will occur to some extent, which induces dimerization and even polymerization, as discussed in previous sections. The

formation of mixed species may not be advantageous in enzymatic

reactions. In addition, these M(II) ions have the tendency to form a hydroxo complex (Figure 4.14), which will cause "self-activation dephosphorylation" (Sigel et a l., 1984). Some trivalent metal ions, such as Sc(III), La(III), and Lu(III), do not interact with the adenine moiety, but they form predominantly a, B, y-tridentate with

ATP in 1:2 stoichiometry (Chapter V), which may result in large steric hindrance to the phosphate acceptor. In addition, these

M(III)-ATP complexes seem to promote dephosphorylation of ATP, and hydrolyze to ADP and AMP spontaneously. Some metal ions, such as

Cr(III), Co(III), and Rh(III), have very slow exchange rates, which is inadequate since transfer of the y-phosphoryl group to the acceptor also requires simultaneous departure of the metal ion from the y-phosphate. The very "hard" Lewis acids, such as Al(III) and

Be(II), prefer the strong base OH” over nucleotides as the ligand at neutral or siightly.basic pH (Bock & Ash, 1980; Karlik et a l., 1983).

The remaining four group 2A metal ions, Mg(II), Ca(II), Sr(II), and Ba(II), all interact with a- and B-phosphates of ADP, while their 83 complexes with ATP are most likely mixtures of a, B, y-tridentate and

R, y-bidentate (Section 4.2). No direct M(11)-N 7 interaction occurs. Among the four metal ions, Mg(II) forms the most stable complexes with ADP (Taqui Khan & Martel 1, 1962) and with ATP (Taqui

Khan & Martel 1 , 1966), which is an essential factor in orienting the phosphate moiety in the active site of enzymes. There is little tendency for Mg(II) to form a hydroxo complex (Figure 4.14) as other divalent transition metal ions. The Mg(II) ion is also the best

Lewis acid among the four, which could function as an electrophilic center and assist the transfer of the Y-phosphoryl group. The rate constants for dissociation of Mg(ll) from ADP and ATP are both larye

(7000 s"1) and the association rate constants is even larger

(Pecoraro et al., 1984), which allows rapid interconversion between

Mg(II)ATP and Mg(II)ADP in the enzyme active site. Figure 4.15 Proposed structure of the reactive [M 2 (ATP)]2 (0 H)~ dimer, which occurs'1 n low concentrations during the metal ion promoted dephosphorylation of ATP. The Intramolecular attack of OH" is indicated on the right side. CHAPTER V

THE INTERACTION OF M (III) WITH ATP

In this chapter, 3 1 P, 1 7 0, and NMR will be employed to study the stoichiometry as well as the coordinating sites of Sc(111)•ATP,

La(111) -ATP, and Lu(111) »ATP complexes.

5.1 31P NMR Properties of H(III)«ATP Complexes

Figure 5.1,A shows the 3*P NMR spectrum of 10 mM ATP, pH 8.0,

free in solution. As illustrated in 5.1,B, a new set of peaks

corresponding to the metal bound complex appears at - 8 . 8 , - 1 1 . 8 , and

-19.9 ppm after Sc(III) is added. It is clear that the exchange rate Ol between free and bound ATP is slow relative to the P NMR time scale. The intensities of the new peaks increase linearly as the ratio of [Sc(III)]/[ATP] varies from 0 to 0.5. The disappearance of the free ATP signals at the ratio of [Sc(III)]:[ATP] = 0.5 (spectrum

E) indicates that at this ratio all the ATP is present as metal bound form. Therefore, the stoichiometry of 1:2. (i.e. the formation of a

Sc( 1 1 1 )(ATP)g complex) is obtained. The assignments of the Pa and P^ of Sc(III)(ATP ) 2 were based on the 3 1 P(1 7 0) signals of [Y- 1 7 0 3 ]ATP.

As shown in Figure 5.2, upon binding of Sc(III), the *70 quenched P^ shifts to -8 . 8 ppm, and a 3 1 P(1 7 0) "line sharpening effect" occurs to 85 86

this resonance. However, the nonlabeled Pa shifted to -11.8 ppm.

The31P NMR chemical shifts and coupling constants, together with the

parameters of other M(111)ATP complexes in this study, are summarized

in Table 5.1.

Figure 5.3 shows the 3*P NMR spectra for the titration of ATP

with varying concentrations of Lu(III). Upon binding of Lu(III), PQ

and Pp shift downfield, Py, on the other hand, shifts upfield. The

chemical shift changes of the 31P resonances are expected to be a

function of the ratio of [Lu(III)]/[ATP]. The binding stoichiometry

was therefore determined by nonlinear least square fittings based on

the following equilibrium:

nATP + Lu(III) *=*= Lu(ATP)n

The curves generated for Pa, PR and P^ resonances yield n values

(stoichiometry) of 2.28, 1.89 and 1.90 respectively. (See Table 5.2

and the appendixes in the end of text for the statistical fitting

results.) The plot of the theoretical and experimental shifts vs. the ratio of [Lu(III)]/[ATP] is shown in Figure 5.4 together with a

simulated PR curve assuming a 1:1 stoichiometry. Thus, the formation

of Lu(III)(ATP)g complex is observed. The broadening of the PR

signal at [Lu(III)]/[ATP] < 0.5 shows that the exchange rate between

PR of the free ATP and that of Lu( 1 1 1 )(ATP) 2 is in the intermediate

range on the 3*P NMR time scale. At [Lu(III)]/[ATP]=0.5, PR becomes sharpened again. This indicates again that a single species is present and a 1:2 stoichiometry is observed. A single set of Pa and 87

/ R P' 0.25

0.30

0.40

0.50

0.80

1— 1 I I I I 1 I I I I I -2 - 6 -10 -14 -18 -22 ppm

Figure 5.1 3*P NMR (81.0 MHz) spectra of ATP (10 mM, pH 8.0), with varying concentrations of ScCl-j. Spectral parameters: spectral width 2500 Hz, acquisition time 1.6 sec, 60° pulse, line broadening 2

Hz, 30 + 2°C. The signals P0, Pp, and Py are due to free ATP whereas

Pa't Pq', and Py' are due to complexed ATP. Table 5.1 ^ P NMR Properties of M(III)«ATP Complexes at pH 8.0

ch em ical s h i f t s ex ch an ae couolina const com plex P <*Pa) (AP3) P UP ) P P a P8 Y r a pa Y J a* J 8Y

ATP - 1 0 .6 -2 1 .3 - 5 .7 19.5 1 9 .9

Sc(III)(ATP}2 - 1 1 .8 ( - 1 .2 ) -1 9 .9 (+ 1 .4 ) - 3 .3 ( - 3 .1 ) Si ow si ow slow 17.0 1 8 .7

La(iri)(ATPJ2 -1 1 .1 (-0.5) -18.3 (+ 2 .5 ) - 5 .5 (+0.1) f a s t f a s t f a s t 17.4 1 7 .7

Lu(111)(ATP)2 -10.3 (+0.3) -17.3 (+3.5) -6.0 (-0.3) f a s t i n t . f a s t 15.5 1 7 .2 89

/

X X X 0 -10 -20 ppm

Figure 5.2 NMR spectra showing the assignment of the Py signal of

Sc( III) (ATP)2 » (A)' Cy-^OjJATP, 10 mM; (B) after addition of 5 mM

ScCl0 . Pa X

Jl j V 0.2

0.3 5 jl JV

0.50 1 I

JL 0.10

- 2 0 -1 0 ppm

Figure 5.3 NMR (81.0 MHz) spectra of ATP (10 mM, pH 8.0), with varying concentrations of LuClj. Spectral parameters are the same in Figure 5.1. 91

Table 5.2 Nonlinear least square fitting results for the

stoichiometry of La(III)-ATP and Lu(III)-ATP

p0 pe pt La(111)a 1.98 ± 0.06 1.91 ± 0.07 1.89 ± 0.20

Lu( 111)b 2.28 ± 0.23 1.89 ± 0.10 1.90 ± 0.07

H8 ^ 2 H1 La(III) 2.03 ± 0.16 2.02 + 0.09 2.05 t 0.18

Lu( 111) ,< 0 4 1 ° ‘ 0 4 1 - 75 1 0 ,4 , -8 9 A ° - 33

aBased on an association constant 3 x 10^ M~* (Morrison & Cleland, 1983)

^Based on an association constant 2.2 x lu^ M”* (Morrison & Cleland, 1983) 22.0

20.0

PPM

10.0

0.2 0.4 0.6 0.0 1 .0 1 .2

ILu(III)]/[ATP]

O 1 Figure 5.4 The plot of theoretical and experimental J P chemical

shifts of ATP (10 mM) as a function of [Lu(III)]/[ATP]. The dashed

Line (— ) represents the Pp of the simulated Ln(111)ATP (1:1

stoichiometry). u

MMfc. A'lP

0.25

jL_ 0.30

0.40

0.50

0.00

0.90

10 -JO ppm

Figure 5.5 31P NMR-(81.0 MHz) spectra of ATP (10 mM, pH 8.0) with varying concentrations of LaClg. Spectral parameters are the same in Figure 5.1. 94

22.0

20.0

18.0

PPM

1 1 .0

6.0

0.2 0.4 0.6 0.8 1 .0 1.2

[La(III)]/[ATP]

Figure 5.6 The plot of 31P chemical shifts of ATP (10 mM) as a

function of [La(III)]/[ATP]. 95

PY peaks representing the averaging of bound and free species throughout the titration course, illustrates fast exchange rate.

Figure 5.5 shows the 31P NMR spectra of ATP as the concentration of La(III) is varied. The plot of 3^P chemical shifts as a function of La(III)/ATP, and the data fitting are shown in Figure 5.6, and

Table 5.2, respectively. Again, the plateau of the chemical shifts beyond La(III)/ATP = 0.5 and the fitting results indicate clearly a stoichiometry of 1:2 between La( 111) and ATP. Furthermore, a single set of peaks through the titration course indicates a fast exchange rate between free and bound ATP.

5.2 170 NMR Properties of M(III)»ATP Complexes

The effects of Sc(III) binding at the phosphate moiety of ATP are illustrated by *70 NMR line broadening effect. As shown in

Figure 5.7, the addition of Sc(111) to ta-^7023ATP causes the appearance of a second downfield signal. This indicates a slow exchange between free and bound ATP on the *70 NMR time scale. Because of the intrinsicaly broad *70 NMR signal, these two peaks do not resolve to the extent that the 3*P NMR peaks do. However, the two components can be identified without artifical deconvolution. The sharp component at 93.3 ppm represents free ATP, the broad component at 102 ppm is metal bound ATP. When [Sc(III)]/[ATP] = 0.53, only the broad component representing the Sc(III)(ATP ) 2 complex is observed. This observation is consistent with the formation of a 1 : 2 complex.

Similar *70 NMR properties: line broadening, downfield shifting, and slow exchange have been observed for the interaction of Sc(III) with 96 i2 io

Sc(UJL)/l> - 0 2]ATP

0.8

0.53

0.35

0.2

200 100 - 1 0 00 - 2 0 0 ppm

Figure 5.7 170 NMR spectra (40.68 MHz) of [a-17023ATP (10 mM in 17 0- depleted water, pH 8.0) with varying concentrations of ScCl3.

Spectral parameters: spectral width 20,000 Hz, acquisition time

102.4 msec, receiver gate 30 usee, line broadening 50 Hz. The Tj inversion-recovery experiment was used for partial suppression of the solvent signal (which has longer Tj than the sample signal), with

180° pulse, 90° pulse, and t as 52 ysec, 26 gsec, and 5 msec, respectively. The delay between acquisitions was 20 msec. 97

H ,0

Sc(m)/ / [ p - ,7o2] ATP

V.3 I y

- 1 0 0 PPM

Figure 5.8 ^ 0 NMR spectra (40.68 MHz) of [B-^02]ATP with varying concentrations of SCCI 3 . The sample and spectral conditions are the same as Figure 5.7. 98

h 2o

] ATP

i—r i — i— i— r i—r T T T 200 100 0 -100 -200 PPM

Figure 5.9 ^ 0 NMR spectra (40.68 MHz) of Cy-^^JA T P with varying concentrations of SCCI 3 . The sample and spectral conditions are the same as Figure 3.7. 99

[B-1 7 02] and [y- 1 7 03]ATP (Fig. 5.8 and 5.9 respectively). The existence

of a single signal beyond [Sc(III)]/[ATP] =0.5 again indicates the

formation of 1:2 complex. The *70 NMR parameters for Sc(111)(ATP)2»

La(III)(ATP)2» and Lu(III)(ATP ) 2 are listed in Table 5.3.

The increase of AO in Figure 5.7, 8 , 9 could be due to the

increasing n, and/or the increasing xr caused by the coordination of

Sc(III). The detailed mechanism needs further investigation. For

Sc(III)(ATP)2 * the R values of a - , B -, and y-phosphates are 2.4, 2.3, and 4.8, respectively. Although the line broadening effect of y-*70

is twice as large, the AO5 values of these a - , B-, and y - 1 7 0 are within + % of one another and a relatively small AOf of y - * 7 0 may

result in a greater R value. It is most reasonable to conclude that

Sc(III)(ATP) 2 exists predominantly as a, 0 , y-tridentate.

The coordinating effect of La (111) to [y- 1 7 03]ATP is shown in

Figure 5.10. The successive addition of metal ions causes *70 NMR signals to broaden and shift downfield. The appearance of only one signal through the titraton illustrates that the exchange rate is fast relative to the *70 NMR time scale, and this signal is the average of free and bound [y-* 7 03 ]ATP. In Figure 5.11, the line widths(AO) of [a- 1 7 02 ]ATP, [B-1 7 02]ATP and [y- 1 7 03]ATP are plotted as a function of the [La(III)]/[ATP] ratio. The corresponding chemical shifts (60) are plotted in Figure 5.12. Both AO and 60 change linearly up to [La(III)]/[ATP] = 0.5 and seems unchanged after this ratio. This is in accordance with a stoichiometry of La(111)/ATP =

0.5 as suggested by ^*P NMR data.

The data quality here is limited by the experimental erorr Table 5.3 Summary of ^ 0 NMR Results

' AOf o r APb , Hz3 R values 60, ppm*’

a 8 Y a S Y a B Y

ATP 480 430 290 9 3 .3 100.3 1 0 b .5

Sc(III)(ATP)2 1620 1420 1680 2 .4 2 .3 4 .8 102(+9) lU6(+6) 118(+13)

La

Lu(III)(ATP)2 1530 1550 1680 2.2 2 .6 4 .8 99(+ 6) 105(+5) 114(+9)

aThe estimated error is ± 55 for AOf and ± 105 for A0b.

^The estimated error is ± 0.5 ppm for free ATP and £ 1.5 ppm for complexed ATP. The numbers in parentheses are changes from free ATP.

o 101

L o u m /lr - ,7o 3 ] ATP

0.5

0.35

0.2

200 100 0 - 1 0 0 — 200 ppm

Figure 5.10 ^0 NMR spectra (40.68 MHz) of [y-^OgjATP (10 mM in

^O-depleted water* pH 8.0) with varying concentrations of LaClg.

Spectral parameters are the same as Figure 5.7. 102

1400 ‘

[a- 1 7 02] ATP

1000

H z

600

200 0.2 0.4 0.6 0.8 1 .0 1 .2

[L b (U I) J/IATPJ

Figure 5.11 170 NMR line widths of [a-1702], [B-1702] and [y-

17 03]ATP (10 mM) with varying concentrations of LaC13. 103

1 20

1 10

PPM

100

90

— ,—

0.2 0.6 0.0 1.0 1 . 2

l L a ( I I I ) J/[ATP]

Figure 5.12 *^0 NMR chemical shifts of [a-^Og], and [y-

*^3]ATP (io mM) with varying concentrations of LaCl-j. 104

Lu(in)/r - ,7a]ATPC

200 100 0 - 1 0 0 - 2 0 0 PPM

Figure 5.13 *70 NMR spectra (40.68 MHz) of [a- 1 7 02]ATP (10 mM in

^0-depleted water, pH 8.0) with varying concentrations of LuClg. 105 resulting from the broad ^ 0 NMR signal. However, if one considers the chances of the same systematic error occuring in three separate experiments with three different samples and at three various ratios, the conclusion about stoichiometry seems reasonable. Furthermore, the 1:2 stoichiometry has been well established by NMR.

Figure 5.13 shows the effect of Lu(III) on the 170 NMR properties of [« -^ 0 2 ]ATP. The changes of chemical shifts and line widths are similar to those of La( 1 II)(ATP)g complex.

As shown in Table 5.3, The R values for the a-, B-, and y-^0 of

Lu(III)(ATP) 2 are comparable to those of Sc( 1 1 1 )(ATP)2 - It is likely that Lu(III)(ATP) 2 is also a, $, y-tridentate. The AO^ and R values of La( III)(ATP ) 2 are smaller than those of Sc(III)(ATP) 2 and

Lu( 1 1 1 ) (ATP) 2 - The AO^ of y- ^ 0 is substantially larger than that of a- and B-^0 in this case. The data may be interpreted as either a tridentate with a stronger y coordination, or a mixture of a, 0 , y- tridentate (ca. 75%) and y-monodentate (ca. 25%), in the case of

La (111)(ATP)2 •

5.3 1H NMR Properties of M(II1)ATP Complexes

Figure 5.14 shows the *H NMR spectra for the aromatic protons of

ATP free and with varying concentrations of Sc(III). The coordination of Sc( 111) induces the appearance of a new set of peaks. It is obvious that the exchange rate for the free and bound ATP is slow. The chemical shifts and coupling constants are listed in Table 5.4. The small shift of H2' (0.062 ppm) sets the upper limit of the exchange 2 S cin D /A T P

H8 h ; 1 I 0 B jJl_ k 0.20

-A Jl 040

Jl IL* 50

L _X . A 080

— I I I I______I______I______I------1 L. 9.0 8.0 7.0 6.0 5.0 ppm

Figure 5.14 *H NMR (200 MHz) spectra of ATP (20 mM in D 2 0, pD 8 . 0 ) with varying concentrations of ScClg. Spectral parameters: spectral width 2000 Hz, acquisition time 4.1 sec, line broadening 0.4 Hz, 30 ±

2°C. H2, Hg and H^ are signals of free ATP. Table 5.4 Proton NMR Results of Metal (III)*ATP Complexes at pD 8.0

coupling

chemical snifts. oom ______const (Hz)

complex H8 H2 HI' H2' H3' H4' H5'5" J 1 '2 '

ATP (8.545) (8.248) (6.136) (4.812) (4.639) (4.390) (4.24) (6.1)

Sc(III)(ATP)2 -0.445 0 -0.156 -0.U62 -0.1U9 0

La(III)(ATP)2 -0.175 -0.158 -0.136 -U.122 -0.099 -0.04 +0.03 -0 .8

Lu (III)(ATP)2 -0.225 -0.178 -0.166 -0.142 -0.129 -0.04 +0.06 -0.5 108

8.5

PPM

5.5

0.2 0.4 0.6 0.8 1 .0 1.2

[La(III)]/[ATP]

Figure 5.15 chemical shifts of Hg, H2, and of ATP (20 mM, pD

8 . 0 ) with varying concentrations of LaC^. 109

B .5

.0

6.0

5 .5

1 .2 2 0.6 0 .B 1 .0

[Lu (III)]/[ATP]

Figure 5.16 XH chemical shifts of H8, H2, and H f of ATP (20 mM, pD

8.0) with varying concentrations of LuClg. 110

rate of Sc(III)(ATP )2 at 12 s"*. It seems that H g of the adenine ring shifts 0.46 ppm upfield; H 2 remains intact. An alternative interpretation is that Hg is shifted to where H2 was, and H2 is shifted to 8.10 ppm. The ribose protons are also shifted upfield to varying degrees, as summarized in Table 5.4. The upfield shifting is best explained by the ring current effect caused by the base stacking from the base rings of the two molecules of ATP in the ternary complex of Sc(III)(ATP)2.

In the previous chapter, Hg downfield shift has been used to illustrate the direct interaction of Ny with metal ions. Sc(1II) is, therefore, expected to bind predominantly to the PQ, Pg, of two

ATP molecules in an octahedral manner, but not coordinate the base ring. The upfield shift of Hg of Sc(111)(ATP ) 2 may also suggest the absence of a M(III)«Ny interaction.

La(III) and Lu(III) also induce most protons of ATP to shift upfield, as shown in Table 5.4. The exchange rates for the two

M(III)-ATP complexes are fast relative to the *H NMR time scale.

Thus, the binding stoichiometry may also be obtained by the fitting program used in Section 5.1. The results are shown in Table 5.2. In

Figure 5.15 and 5.16, the chemical shifts of aromatic protons are plotted as functions of [La(III)]/[ATP] and [Lu(111)]/[ATP] ratios; the changes level off at [M(III)]/[ATP] = 0.5, in agreement with ^ 0 and ^ P NMR results. Ill

5.4 Discussion

5.4.1 Stoichiometry of ATP Complexes of S c(lII), L a(III), Lu(III)

For Sc(III)(ATP) 2 » all three NMR techniques exhibit the slow exchange; the stoichiometry of 1 : 2 is determined by the disappearance of free ATP signals at [Sc(III)]/[ATP] = 0.5. The two lanthanide ions, La(III) and Lu(III), induce fast exchange between free and bound ligands in all three NMR spectra. The plateau of the NMR titration curves beyond [M(III)]/[ATP] = 0.5 indicates a stoichiometry of 1 : 2 .

The formation of 1:2 Ln(111) -ATP complexes at millimolar ATP concentrations is supported by several independent studies: Eu(III) luminescence and Pr(III) paramagnetic effect on NMR relaxation (Eads et al., 1984); EPR spin-echo of Ce(III), Nd(III), Er(III), and

Yb(III) complexed to ATP (Shimizu et a l., 1979, 1983). Recently, the formation of 1 : 2 1 anthanide»triphosphate complex was illustrated by the multi nuclear NMR shift and relaxation rate measurements

(Nieuwenhuizen et a l., 1985).

The stoichiometry is believed to be dependent on the ratio of

[M(III)]/[ATP] as well as the concentration range. At pH 8.0 and with a higher ratio of M(III)/ATP, (i.e. beyond [M(III)]/CATP] =

0.5), due to the net "-5" charge on the M(111)(ATP )2 complex, the

M(III) added is very likely to coordinate with the 1:2 complexes.

This can occur by coordinating a M(III) with a single M(111) (ATP ) 2 to form a 2 : 2 quarternary complex and/or by bridging with two ternary complexes to form a 3:4 structure. High order oligomers may be present if more metal ions are introduced. The addition of M(III) to 112

the M(1 1 1 )(ATP)2 In this study caused slight broadening of the NMR

signals and enhanced the decomposition of ATP. However, there is a lack of evidence for other stoichiometry. More complicated structures, which involve more than one metal ion, are not strictly

ruled out by the present data.

1:1 stoichiometry of Ln(III)-ATP has been suggested in the enzyme kinetic experiments to study the dissociation constants of these complexes (Morrison & Cleland, 1983; Morrison & Cleland, 1980;

Viola et a l., 1980). In their experimental conditions (low concentrations M(III), excess Mg(11) over ATP), it is likely that the formation of MgATP lowers the concentration of free ATP, thus, favoring the formation of 1:1 complexes. The dissociation constant for Eu(III)( ATP) 2 Eu (111) ATP + ATP has been suggested as 300 + 50 pM (Eads et a l., 1984). When ATP is present at a low concentration range (pM) and the ratio of [M(III)]/[ATP] is low, the predominant stoichiometry could be 1 : 1 .

5.4.2 Structures of M(III)(ATP ) 2

Viola et al. (1980) suggested that the binding strength of

M(111)ATP complexes with hexokinase might relate to the different proportions of fl, y-bidentate isomers present in solution, which could in turn relate to the ionic radius with 0.88 A being the critical size. On the basis of their suggestion, La(III)ATP (ionic radius 1.06 A, =. 174 + 8 pM) should have a higher percentage of tridentate, whereas Sc(III)ATP (ionic radius 0.73 A, = 8.0 + 2.9 Table 5.5 Ln(III)-P Distance (A)a

Ionic radii Distance

Ln (A) Ln-Ptt»Y Ln-Pe

Nd 1 . 0 3.44 3.52

Eu 0.95 3.06 3.13

Tm 0.87 3.59 3.43

Yb 0 . 8 6 3.57 3.38

aAdapted from Nieuwenhuizen et al, 1985. 114

yM) and Lu(1II)ATP (ionic radius 0.85 A, = 0.8 yM) should have a

higher percentage of the 8 , y-bidentate. Our conclusion that

tridentate is the predominant structure of these three M( 1 1 1 )(ATP) 2

complexes, regardless of the ionic radius is not fully compatible

with the interpretation of Viola et a l., but is supported by the

report of Nieuwenhuizen et al. (1985). As shown in Table 5.5, the

distance between several lanthanides and the P„ and P« of a» Y p triphosphate is unrelated to the ionic radii. However, one

possibility that cannot be rultd out is that bidentate exists in a

small percentage beyond detection by 31p NMR, but still varies with

ionic radii.

Figure 5.17 shows the most reasonable structure for

Sc(III)(ATP)2 * It appears that the six phosphates from the two ATP

molecules occupy the six coordination sites of an octahedral

structure, with the two adenine rings partially stacked. The

structures of La(III)(ATP ) 2 and Lu(III)(ATP )2 may be similar to that

of Sc(111)(ATP) 2 » but it is possible for the lanthanide ions to have

higher coordination numbers (Huheey, 1978) by coordinating additional water ligands.

There are four possible diastereomers for the ot, 8 , y-tridentate

M(III)(ATP)2 - These diastereomers should have distinctly different

^ P and 170 chemical shifts, based on the observation of Co(111) complexed to ADP and ATP (Huang & Tsai, 1982; Sammons et a l., 1983;

Cornelius et a l., L977). However, only one set of ^*P NMR and *70

NMR signals was observed for Sc(III)(ATP)2, even though its exchange Figure 5.17 The proposed structure of Sc(III)(ATP )2 116

rate is slow in the NMR time scale (exchange rate < 12 s - 1 at

30°C). Two possible explanations are: (i) only one diastereomer is formed specifically, or (ii) the exchange between diastereomers is a distinct process from the exchange between free and bound ATP and is fast.

Explanation (i) is chemically unlikely in the absence of enzyme. Explanation (ii) is not only consistent with all the data, but also precedented by Co(III) (NH 3 )4 ADP (Sammons et a l., 1983). By use of stereospecifically labeled A and A isomers of CoUllMNHgJ^Sp)

-[a-^0j]ADP, the interconversion between the two diastereomers has been shown to occur faster than dissociation of the complexes, though slower than the ^ P and 170 NMR time scales.

It is not known whether such a rapid intramolecular interconver­ sion also occurs between tridentate, bidentate, and monodentate isomers. If not, the Sc(111)(ATP )2 should consist of a, B, y- tridentate as the only species, since the complex is in slow exchange

(with free ATP) on the NMR time scale, and only a single set of spectra is observed in *H, ^*P, and ^ 0 NMR. On the other hand, if such an interconversion is a fast process, the structure of

Sc(111)(ATP)g could actually be a mixture of several species, e.g., tridentate, bidentate, and monodentate, with tridentate being the predominant one. In any case, the spectral data represents the average of several different species (diastereomers and/or positional isomers); the "exchange rate" represents the rate of exchange between free ATP and an average of several species of complexed ATP. CHAPTER VI

31P NMR STUDIES OF BINDING OF AMP AND ATP TO ADENYLATE KINASE

The stoichiometry of [AK]:[ATP] and the crossbinding of substrates (i.e. AMP binding to the MgATP site, ATP binding to the

AMP site) are the subjects of this chapter. By using *7 0-labeled as well as nonlabeled nucleotides, we address these issues with 3*P MMR experiments. In Section 6.1, tritration of ATP with AK indicates a stoichiometry of 1:1. In Section 6.2, the crossbinding of AMP at the

MgATP site, based on a broadened AMP signal at the ratio of

[AMP]:[AK] = 1:0.2, is suggested. In Section 6.3, 3 1 P(1 7 0) experiments are presented to argue against the possibility that both

ATP and AMP bind to the same site.

6.1 The Stoichiometry of AK:ATP

Both 1:1 and 1:2 stoichiometry of [AK]:[ATP] have been proposed by different research groups with various methods (see Section

01 1.3). In order to resolve this controversy, the P NMR titration experiments of ATP with varying concentrations of AK are performed.

Although the 3*P chemical shifts of ATP are unaffected by the binding of AK (Nageswara Rao & Cohn, 1978), the changes in Jap and

117 118

JpY, as well as the increase in the linewidths (AP) of the signals during the titration course are used to determine the stoichiometry of ATP binding to AK. Figure 6.1 shows the PQ signals of ATP free and bound to varying concentrations of AK. The data on the chemical shifts, coupling constants and linewidths are included in Table

6.1. Upon addition of AK, as shown in Figure 6.1, Jaf? decreases gradually from 19.6 Hz to 18.2 Hz until [AKj/LATP] reaches 1.0. The plots of the changes of J a 0 and as a function of the ratio of

[AK]/[ATP] are shown in Figure 6.2. The measurement of linewidths,

APa and APy also show a linear increase, which is shown in Figure

6.3. Based on the assumed association constant (3 x 10^ M"*, Price et a l, 1973), the stoichiometry was determined by nonlinear least square data fitting as described in Section 3.8. The results are shown in Table 6.2 and in the appendixes. Both coupling constants and linewidths show approximately linear changes from [AK]/[ATPJ = 0

- 1.0. The curves flatten after [AK]/[ATP] = 1.0, which indicates clearly that the stoichiometry of [AK]:[ATP] is 1:1.

Table 6.2 Nonlinear Least Square Fitting Results for the

Stoichiometry of AK»

^aB

1.04 + 0.17 0.98 t 0.06 1.00 ± 0.02 1.00 ± 0.03 119

[AK]/[ATP]

.4 J 1 .3

■ ■ ■____ i— i— i— i— i— i— *— i— >— ■— > 5 Hz

Figure 6.1 NMR signals of PQ of ATP free, anti bound, to varying concentrations of AK at pH 7.8 and 10°C. Spectral parameters: spectral width 5UUU Hz, acquisition time 1.6 s , 6 U° pulse, line broadening 2 Hz, 4000~8000 scans. The solution of A contained 2.3 mM

ATP, 70 mM triethanolamine-HCl, 65 mM KC1, 1.5 mM DTE, and 20% 0 2 0.

A solution of 3.0 mM AK was added to solution A to attain the molar ratios of [ATP]:[AK] shown in the figure. Table 6.1 ^lp NMR Parameters of Free and Enzyme-Bound Substrates

of AK at 10°C, pH 7.8

Comolexes S ( p pin 'I J (HZ) aP (Hz)

ATP AMP ATP ATP AMP

oB By free subtrates -10.7 -21.5 -6.0 3 .9 1 9 .6 1 9 .9 3.5 3.5 4.0

AK.ATP ■10.7 -21.4 ■ 6.0 1 8 .2 2 1 .5 6 .8 5.2

AK-AMP 4 .1 5 .0 120 121

22.0

20.0

Hz

0.4 0.8 1 .2 1 .6 2.0

[ AK] / [ ATP]

Figure 6.2 The calcu1ated(—-) and experimental 31p_31p spin-spln

coupling constants JaB and J Ry as a function of [AK]/[ATP]. The data

for [AK]/[ATP] = 2.0 were obtained from AK»ATP»AMP ternary complex.

The theoretical val.'ues were calculated based on 1:1 stoichiometry and

a binding constant of 3 x 10 4 M_1 (Price et al., 1973). 122

o

0

.0

.0

0.4 0.8 1 .2 t . 6 2.0

tAK]/ [ATP]

Figure 6.3 The calcu lated — ) and experimental APa(0), AP^(X) (the linewidths of Pa and Py) as a function of [AK]/[ATP]. The data for

[AK]/[ATP] = 2.0 were obtained from AK*ATP*AMP ternary complex. The theoretical value were calculated based on 1 : 1 stoichiometry and a binding constant of 3 x 10^ M"* (Price et a l., 1973). 123

6.2 The Crossbinding of AMP

The binding of AMP to the MgATP site has been suggested by several studies (Section 1.3). It seems that the most direct method to tackle this question is equilibrium dialysis. However, the binding study of AMP by equilibrium dialysis cannot exclude the possibility that AMP binds to the MgATP site (Ito et al., 1980). In this section, a more direct ^P NMR evidence is presented which

supports the crossbinding of AMP. We titrated AMP with varying

O 1 concentrations of porcine AK, and observed the changes in JAP NMR line widths.

The data of Table 6 .3,A corresponds to the ^ P NMR signal of AMP free in solution, having a line width of 4 Hz. 6.3, B was obtained after 0.2 eq. of AK had been added. The line width of this partially bound AMP increases to 20 Hz. When more AK was added, as shown in

6.3,C and D, the AMP signal was further narrowed. 6.3,E representing a stoichiometry of [AK]/[AMP] = 1:1.1, shows a 5 Hz linewidth.

Two possible interpretations for the broadening ^ P AMP signal during the titraion course are: (i) the exchange rate between free and bound AMP is in the intermediate range, and (ii) AMP is able to bind to both sites and the exchange rate is in the intermediate range. Based on the following reasons, the latter seems to be more reasonable than the former. Firstly, if (i) were true, at AMP:AK =

0.5, half of the AMP would appear as free and half as enzyme bound, then the broadest peak would occur at this ratio instead of 0 . 2 .

Secondly, it has been shown that ADP can undergo exchange between the 124

Table 6.3 31P NMR(202.5 Hz) results of AMP titrated with varying amounts of AK.

Experiment AMP:AK

A 1:0 4 B 1:0.2 20 C 1:0.5 10 D 1:0.8 5 E 1:1.1 5

Spectral parameters: spectral width 10,000 Hz, acquisition time 0.8 sec, 60° pulse, LB = 0.5 Hz, 1000 scans. Sample conditions were the same as in Figure 6.1. two sites at intermediate rate on the NMR time scale (Nageswara et a l.,1978; Vasavada et a l.,1984), whereas the exchange between free and bound ATP is fast on the NMR time scale (Section 5.1). Thus, the broadened AMP signal is more likely due to the exchange between two sites but not to the on-off from the enzyme. It seems that the most reasonable interpretation for Experiments 6.3, A - E is as follows: when the concentration of AK is much lower than that of AMP,

(i.e.,AMP:AK = 0.2), there are not enough AMP sites available for the specific binding of AMP. As a consequence, AMP is less discriminating between the two sites and gives an exchange-broadened signal. When more enzyme is involved (6.3 C,D), AMP has a preference to choose its own site, resulting in narrower signals. When the concentration of

AK exceeds that of AMP as shown in Experiment 6.3,E, AMP should bind exclusively to the AMP site.

6.3 Differentiation Between the ATP and AMP Sites of AK

In Sections 6.1 and 6.2, we have demonstrated the formation of

1:1 AK»ATP complex, as well as the possible binding of AMP to the

MgATP site at a low [AK]/[AMP] ratio. The MgATP site and the AMP site have been identified as residues 1-44 and residues 172-194, respectively, based on the binding properties of peptide fragments containing these residues (Hamada et a l., 1979). The MgATP binding fragment (1-44) binds ATP, MgATP, ADP, MgADP, but not AMP, whereas the AMP binding fragment (172-194) binds AMP and ADP, but not ATP,

MgATP or MgADP. Binding of MgATP to the fragment 1-44 has also been confirmed by NMR studies (Fry et a l., 1985). Based on these 126 studies, it seems reasonable to assume that in the AK»ATP binary complex, ATP binds to the MgATP site at the N-terminal region, whereas in the AK*AMP binary complex AMP binds to the AMP site at the

C-terminal region.

However, the MgATP and AMP sites assigned by Hamada et a l.

(1979) and Fry et al. (1985), based on the peptide fragments, are opposite to those assigned by Pai et a l.(1977), based on an x-ray structural study of the intact AK. In addition, considering the fact that binding of ATP to both MgATP and AMP sites has been proposed by several groups, as reviewed in Section 1.3 and crossbinding of AMP to both sites also occurs, as described in Section 6.2, we cannot completely rule out the possibility that both ATP and AMP bind to the same site (AMP site or MgATP site) of the intact AK at the resting state and in the absence of Mg(II). In this section, we present

31P(170) NMR titration experiments to argue against such a possibility. The results (see 6.3.1) suggest that the Pa of AMP is bound more rigidly than the P 0 of ATP, and implies that they bind to different sites (i.e. ATP should not bind to the AMP site in the

AK*ATP binary complex).

6.3.1 Use of 31p(17q) NMR to Compare the Dynamics of P0 in AK-ATP and AK-AMP.

Figures 6.4 and 6.5 illustrate that the 31p(17o) effect of [a-

^ 0 £]ATP is different from that of [^(^jAMP when these two nucleotides are totally bound to AK. I have discussed the ^ ( ^ o ) effect in detail in Section 2.2. In summary, if AO (the linewidth of 127

[((-"OjjjATP.-AK 1.0:0

1.0 * 0.2

B

1.0: 0.5

1.0 : 1.0 D

1.0 : 1.2

■wMW

-20 ppM

Figure 6.4 31P NMR spectra of [a- 1 7 02]ATP free and titrated with varying amounts of AK at pH 7.8 and 10°C. Spectral parameters: spectral width 5000 Hz, acquisition time 1.6 s, 60° pulse, line broadening 2 Hz, 4000-8000 scans. The solution of A contained 3.3 mM

[a-* 7 02 ]ATP, 70 mM tr1ethanol amine-HCl, 65 mM KC1, 1.5 mM DTE, and

20% D20. A solution of 3.3 mM AK was added to solution A to reach the molar ratios of [ATP]:[AK] shown in the figure. The concentrations of ATP and AK in spectrum E were 1.53 and 1.9 mM, respectively. 128

AMP«AK

1.0*0

1.0 : 0.2

.0*0.4

1.0 * 0.6

1.0 * 0 .8

1.0 * 1.0

1.0 * 1.2

JL I 10 0 -1 0 Chemical Shift (ppm)

Figure 6.5 31P NMR spectra of free [^ o 3 ]AMP(1.9 mM) (A) and

AK'C^t^DAMP (B-G). Spectral parameters were the same as In Figure

6.4, except the number of scans were 4,000-10,000 and the line

broadening was 5 Hz. Sample conditions were similar to Figure 6.4,

except that all samples contained 15 equivalents of EDTA relative to

AMP. After spectrum A was obtained, the sample was lyophlllzed and added to a solution of 2.9 mM AK to reach the ratio of [AMP]:[AK]

shown In B-G. 129 effect 1n detail in Section 2.2. In summary, if AO (the linewidth of

1?0 NMR signal) increases due to binding to the enzyme, AP (the line width of 3*P NMR signal) should decrease. As a result, a "line sharpening effect" in 31P NMR can be observed.

In Figure 6 .4,A, the Pa resonance consists of two components.

The integration spreading over a wide range (ca. 400 Hz) indicates the 31P(170) signal. The sharp component is due to non-^0 species. Upon binding to AK, the broad component sharpens and to some extent merges with the sharp component. Consequently, the integration in spectrum E covers only a narrow range. Similar changes have been observed in the binding of * 7 0-labeled ADP to ribonuclease A (Tsai, 1982).

On the other hand, in the titration of [^0g]AMP with adenylate kinase, as shown in Figure 6.5, the integration in spectrum A and G both cover a wide range (ca.600 Hz), indicating the lack of line sharpening in the AK»[^ q,j]/\mp complex (the changes in the titration process B-F will be discussed in the next paragraph). The lack of clear line sharpening effects in AK»[^03]AMP cannot be due to lack of binding since changes are observed in B-F. Rather, it resembles the cases of arginine kinase*ADP (Sammons et a l., 1983) and phosphoglucomutase»substrate complexes (Rhyu et a l., 1984). Since the latter two enzymes are of higher molecular weight than adenylate kinase and ribonuclease A, the results in this study might imply that the Tr of the Pa of AMP is larger than that of the Pa of ATP in the active site of AK, and that the broad component in spectrum G is due 130

31p_160 31p_170 Mi xed

AMP free

(0 % enz.)

AMP in AMP site

(>100% enz., AK»AMP

binary complex

AMP in MgATP site

(imaginary)

AMP exchanging

(20 ~ 40% enz.)

Figure 6 . 6 Simulated ^ P NMR spectra of ^ 0 labeled and regular AMP

bound to AK. a. See Fig. 6.5, A; b. Fig. 6.5, G; c. Table 6.3, E; d. Table 6.3, B; e. Fig. 6.5, B, C. 131

to dipolar instead of quadrupolar effect, as discussed in the last

paragraph of Section 2.4.

Based on the above interpretation, the 3 *P(^0) broad component

in spectrum A is due to the quadrupolar effect, whereas the

broadeners in spectrum G is due to dipolar interaction. An

unanswered question is why the 3 *P(^ 0 ) component seems to be

"sharpened" in spectra B and C, and gradually broadened again from D to G. Since it is postulated from Section 6.2 that AMP exchanges

between the AMP site and the MgATP site under the conditions of B and

C, we think that the "line narrowing effect" of the 3 1 P(^0)

component in B and C is due to that the dynamics of AMP at the MgATP

site is less rigid than that at the AMP site. Although we are unable to simulate or calculate the spectra quantitatively, the line shapes of spectra A-G in Figure 6.5 are qualitatively illustrated in Figure

6.6.

6.3.2 Other Evidence Against Binding of ATP to the AMP Site

Previous ^ 0 NMR studies (Wisner et a l., 1985) have shown that the 170 NMR linewidths of [a-*^]-, and [y-^^JATP increase upon titration with a small percentage of adenylate kinase, whereas the AO of [^Og]AMP is insensitive to the addition of enzyme. It seems that the binding of ^0-labeled ATP is in the "fast exchange limit", whereas that of [^OgDAMP is in the "slow exchange 132

TIME (hours) J____ I

V«il| IV

VIII

Figure 6.7 ^ P NMR spectra of MgATP in the presence of adenylate

kinase as a function of time. The sample contained 20 mM ATP, 80 mM

MgSO^j and 4000 mU/ml adenylate kinase at 30°C. Peak assignments

are: I, y-phosphate of ATP; II, 8 -phosphate of ADP; IV, a-phosphate

of ATP; V, a-phosphate of S^AdoP^; VI, 8 -phosphate of ATP; VII, 8 -

phosphate of 5"AdoP/j; and VIII, y-phosphate of 5"-AdoP/|. (Adapted

from Kupriyanov et a l., 1986.) 133 limit". These results directly support the assymetry of the two sites in binding of substrates.

Recently, a report from Kupriyanov et al.(1986) strongly questioned that ATP binds to the AMP site. As shown in Figure 6.7,

24 hours incubation of MgATP with AK resulted in the formation of adenosine tetraphosphate (5^-AdoP^). The resonance VIII (-21.0 ppm) representing the y-phosphate of this compound is very likely correspondent to the upfield Pg in Figure 1.1,D which had been assigned as ATP binding to the AMP site. This evidence argues against the interpretation of Nageswara Rao et al. (1978) and supports our observation that ATP binds only at the ATP site.

6.4 Discussion

6.4.1 Summary of Results

01 By observing the changes of J1P NMR parameters, the stoichiometry of the nucleotides binding to porcine adenylate kinase has been determined. Furthermore, the specificity of these binding sites has been suggested. The evidence for these points is provided by the following findings as discussed in detail in the previous sections: (1) ATP binds only at the MgATP site, (ii) The stoichiometry of [AK]/[ATP] is 1.0. (iii) At lower ratios of

[AK]/[AMP], AMP binds to both the AMP and the MgATP site. However, at [AK]/[AMP] = 1.0, AMP binds predominantly to the AMP site. The comments on methodology, as well as the impact of this result on AK structure will be mentioned in the next two sections. Table 6.4 3*P chemical shifts (ppm) of free and enzyme-bound

nucleotides at pH 7.0.

Enzyme ADP ATP

P« PB Pa P 8 PY

Arginine kinase 1 0 . 8 5.1 1 1 . 1 21.9 7.3®

Creatine kinase 1 0 . 2 4.8 10.9 2 1 . 0 5.7b

Adenylate kinase 1 1 . 0 2 1 . 8 7.3C

1 0 . 8 7.5 1 1 ; 0 2 1 . 8 7.3d

Phosphoglycerate kinase 11.3 2 2 . 8 6.7e

Pyruvate kinase 10.7 6 . 0 1 1 . 1 21.7 6.4f

a Obtained from Nageswara Rao & Cohn (1977). b Obtained from

Nageswara Rao & Cohn (1981), and pH = 7.8. c Obtained from Section

6.1. d See Table 6.1. e Obtained from Nageswara Rao & Cohn

(1978b). ^ Obtained from Nageswara Rao et al. (1979). 135

6.4.2 Criticism of Methodology and Experiments

01 In this section, I will discuss why and how P NMR spectroscopy was employed to monitor AK»nucleotide complexes.

Proton nucleus is the most sensitive with regard to the signal to noise ratio. However, *H NMR spectra, while reasonably well resolved for small proteins, becomes unresolved for larger enzymes.

The large number of protons present, especially in the aliphatic region, causes a loss in resolution and assignments of individual resonances, the very desired advantage of *H NMR spectroscopy. In addition, *H NMR experiments have to be performed in D20 as a solvent.

The preparation of the sample then becomes critical to the enzyme activity. Based on the above considerations, two questions are raised to the previous ^H NMR studies on AK: ( i ) Were the assignments of each resonance fully convincing? (ii) Did the protein sample possess full activity as in the native enzyme?

The fact that few phosphorus nuclei are present in the system observed by 31P NMR makes the assignment problem much simpler.

Furthermore, the possibility of enzyme denaturation caused by sample preparation is less of a problem. Nhen the E*S complex is observed, then large amounts of enzymes must be used. However, the advantage of doing a 3*P NMR experiment with fully enzyme bound nucleotide is best explained by the fact that the 3*P nuclei are part of the substrate. Thus, one can study the substrate possessing a conformation that resembles the one in the catalytic reaction.

A number of the 31P NMR observations of phosphoryl transfer enzymes has been attributed to enzyme»nucleotide complexes. The 136

requirement of those experiments was that the enzyme concentrations chosen(2 ~ 4 mM) have to be in sufficient excess of the amounts of substrate(l ~ 3 mM) so that 80 ~ 90% of the substrate is in the enzyme bound form. The experiments in my study satisfy the condition above (i.e. when the ratio of [AK]/[nucleotide] >1, with ca.

10"^ M, substrates appear as enzyme bound form). The kinases which have been studied by 3*P nmr with fully bound substrate are arginine

kinase from lobster (Nageswara Rao et a l., 1976.» Nageswara Rao &

Cohn, 1977), rabbit muscle creatine kinase (Nageswara Rao & Cohn,

1981), carp and porcine muscle adenylate kinase (Nageswara Rao et a l., 1978a, Vasavada et a l, 1984), rabbit muscle pyruvate kinase

(Nageswara Rao et a l., 1979), and 3-phosphoglycerate kinase from yeast (Nageswara Rao & Cohn, 1978b).

The 3*P NMR experiments cited from literature together with the 31 studies in the previous section encompass measurements of P NMR parameters such as lineshape, chemical sh ifts, and coupling constants of enzyme bound nucleotides. Table 6.4 summarizes the 31P NMR chemical shift changes of ATP and ADP in these studies. It is obvious that the chemical shifts of some resonances are affected by binding to the enzymes, whereas others are not. The 31p_31p spin- spin coupling constants in the enzyme bound complexes of ATP and ADP are equal to those of the nucleotides free in solution within experimental error, with the exception of ATP in its complex with

AK. Thus, the 31P NMR parameters of kinase-nucleotide complexes do not reveal a systematic change that might signify the similarities in the environment or conformation of the phosphate chain in these complexes. 137

There are two unique aspects which signify this study and

differentiated from the previous 3*P NMR experiments. Firstly, the

titra tio n courses of ATP and AMP with enzyme monitored the changes of

nucleotides stepwisely. Thus, the stoichiometry can be determined

with more accuracy. Secondly, ^lp(17o) method was used to observe

the dynamic properties of nucleotides in the active site.The

consistency of the 3*p-31p Sp-jn-spin coupling constants between free

and enzyme bound nucleotides limits the titration method applied to

other phosphoryl transfer enzyme. However, ^ 1 P(^0) technique can

still be employed to study the substrate binding to other kinases. ^ 0

labeled on different phosphates may be utilized further to pursue the

effects on different portions of the phosphate chain.

One disadvantage of this kind of approach is the requirement for

large amounts of enzyme (5~10 ymole in one experiment). In addition, extra caution must be taken into account since NMR parameter changes involved are usually small.

6.4.3 Implication of the Active Site Structure

The different binding properties of AMP, ADP and ATP asstudied in this research implies that theasynmetry of the binding site may be partially contributed by the recognition function of enzyme active s ite towards different phosphate chains. The asymmetry of the AMP and the MgATP binding site of AK has been demonstrated both in 3*P and NMR experiments. Nageswara Rao et a l. (1978) have identified the 3*P resonances of MgADP and ADP bound at the MgATP and the AMP site. Smith & Mi Idvan (1982) have observed the NMR NOE effect on 138

the C-2 proton of His—36 (located in MgATP site) caused by the

binding of ATP.

The above suggestions are consistent with two possible

structural features of the enzyme, ( i ) Since ATP can only bind to

the HgATP s ite , certain residues in the MgATP binding fragment are

responsible for the interaction with the y-phosphate of ATP. These

residues, however, are absent in the AMP binding pocket, (ii) The

inherent structural fle x ib ility allows the MgATP site to have a lower

specificity. On the other hand, the AMP site is more rigid and

possesses a higher selectivity; this pocket can accommodate only free

AMP and ADP. Either of these two cases above are in agreement with a

1:1 stoichemetry of AK»ATP and AK*AMP binary complexes. However, due to the lack of direct evidence, it is very difficult at this stage to judge which one is correct. It might even be a combination of the two. The results here take on more significance when viewed in the context of existing information from the literature as shown by the following description.

The entire enzyme structure of adenylate kinase has been obtained by x-ray crystallography. The simplified representation is shown in Figure 6 . 8 (Pai et a l., 1977). The MgATP site, has been mapped by *H NMR NOE techniques (Fry et al. 1985). The structure was reported to be accurate within 2 A and is shown in Figure 6.9. The important points of this study are: (i) the potential interactions of

Thr-23 to the a-, B-, and y-phosphates of ATP, (ii) the side chains of Lys-21, Glu-24 can be positioned very close to the a-phosphate and 139

123

119 179

60

69

N

Figure 6 . 8 . Simplified representation of the crystal structure of the entire adenylate kinase molecule showing the location of bound

MgATP as determined by NMR. Helices are indicated by cylinders and

6 -sheets by arrows. The strippled regions are areas that display sequence homology to other proteins. The enzyme structure was obtained from Pai et a l. (1977). 140

to.

iri it

9S

92

Figure 6.9 The proposed MgATP-binding sites of adenylate kinase (A) and the 1-45-residue peptide (B). Enzyme and peptide structures were drawn by using the x-ray coordinates of Sachsenheimer & Schulz

(1977). Residues 10-37 and 90-95 are shown in (A); residues 10-37 are shown in (B) (adapted from Fry et a l., 1985). 141

(iii) Lys-27 can approach the B- and y-phosphates. Therefore, these

residues may play a role in the binding of the terminal phosphate of ATP.

For the AMP pocket, the phosphate of AMP has been assumed to

bind near lysine-172 (Fry et al., 1985). It is noted that the

phosphate of AMP must be oriented near the N-terminal end of an a-

helix (nearby Val-179) in order to approach the MgATP site closely enough. However, there is only one basic residue (Lys-172) in the

168-184 segment, that has potential to intereact electrostatically with the phosphate chain. Accordingly, it is reasonable to assume

that the lack of binding of ATP in the AMP pocket is due to the

shortage of basic residues in this region. Nonetheless, the binding

of unchelated ADP in the AMP site is possibly due to the interaction

of Lys-172 with both the a- and B-phosphates of ADP.

It has been suggested that the hydrophobic pocket surrounding the adenine and ribose moieties of bound ATP accomodate a variety of

base and ribose structures (Noda, 1973; Hamada & Kuby, 1978;

O'Sullivan & Noda, 1968). A number of metal-nucleotide complexes with different structures of phosphate chains have been shown to bind

to adenylate kinase. These complexes include A and A isomers of

Cr(III)-ATP, (Fry et a l., 1985; Dunaway-Mariano & Cl el and, 1980b), Ca

(II), Mn(II), Ba(II), and Co(II) complexes of ATP (Noda, 1958;

Markland & Wadkins, 1966; Su & Russel, 1968; O'Sullivan & Noda,

1968), Mg(11) and Cd(II) complexed to Sp, Rp-ATPaS, Sp, Rp-ATPBS

(Eckstein & Goody, 1976; Tomasselli & Noda, 1983; Kalbitzer et a l.,

1983). These findings concur with the observation in this study that the MgATP site is less rigid, and accommodates metal free adenine 142

nucleotides (including AMP) but with less specificity. Fry et al.

suggested that the ATP molecule is highly extended and cuts across the helix, bend, and strand of a fl-sheet formed by residues 23-27.

This region is likely to possess the flexibility needed for the

various structures of phosphate chains.

Finally, due to the suggested random bi-bi mechanism (Rhoads &

Lowenstein, 1968; Su & Russel, 1968), the rigidty of AMP pocket seems to be inherent but not resulting from a conformational change, after

binding of the cosubstrate. 143

CHAPTER V I1

SOME PRELIMINARY OBSERVATION ON AK-M*ATP

It is well established that kinases utilize MgATP complexes as substrates. In the kinetic studies of kinases, the appropriate dissociation constant for MgATP (0.0143 niM at pH 8.0, Morrison, 1979) was often used to calculate the amount of metal ion and nucleotide to ensure the desired concentration of MgATP complex. Michael is constants for the nucleotide substrates were then usually reported as the MgATP complexes. In such cases, the concentrations of MgATP free in solution were also assumed as the effective substrate concentration in the enzyme active site. However, it has been suggested that creatine kinase (Morrison & O'Sullivan 1965), hexoklnase (Frey et a l., 1974; Mohan & Rechnltz, 1974), and adenylate kinase (Price et a l., 1973) bind unchelated ATP tightly. In the enzyme active site, if ATP is present in a significant amount and functions as a linear competitive inhibitor vs. the MgATP, then the determined values for the maximum velocity and Michael is constant for the catalytic substrate MgATP will be in error. The general equation that describes linear competitive inhibition is

Y - V™x * yi+i/K j) + a 144

where A represents MgATP and I, ATP. Thus, a reduced Vmax (maxiumum

velocity), and a lower Km would be observed. Unfortunately, such a

competition between the nonproductive binding of ATP and the

catalytic binding of MgATP to the enzyme active site (i.e . E»ATP +

MgATP == E«MgATP + ATP) has drawn l i t t l e attention in the past.

In Chapter VI, I indicated that unchelated ATP binds only to

the MgATP site of adenylate kinase. This excludes the existence of

an AK*MgATP»ATP ternary complex. Since the Pp of MgATP appears at

-19 ppm and P 0 of AK*ATP is at -21.4 ppm, an alternative

interpretation of the double Pg resonances in Figure 1.1,D is the

exchange of free MgATP in solution with ATP bound at AK. This

implies that the nonproductive binding of ATP is still observed at a

ratio of [Mg(II)]/[ATP] of 2:1.

In this chapter, three ^*P NMR titration experiments of ATP with

varying amounts of AK and metal ions are designed to observe the

above ambiguity. The results of the fir s t set of experiments (see

Section 7.1) suggest that the nonproductive binding of ATP is

stronger than that of Sc(III)ATP. In the second set of experiments

(Section 7.2), the unchanged NMR data of E-ATP implies that Cd(II)ATP binds weaker than ATP to AK. In the third set of experiments

(Section 7 .3 ), a new Pp peak appeared at -16 ppm beyond

[Mg(II)]/[ATP] =10:1. This implies that the binding of MgATP to AK is observable only at high concentrations of M g (II).

It has to be emphasized that the observation and the interpretation in this chapter are only preliminary at this stage.

The three ^P NMR titration experiments, are not sophisticated enough 145

to resolve unambiguously the above controversy. Detailed studies

such as equilibrium binding, determination of the [Mg(II)] free in

solution , and inhibitory kinetic experiments are needed to support

the current results. Furthermore, if ATP does compete favorably with

MgATP for the active site under catalytic conditions, then there will

be a large impact on the previous kinetic data. Thus, precise and

accurate data is absolutely required to make such a strong statement.

7.1 m r Experiments of AK»Sc(Ill)»ATP

^*P NMR spectra of the equilibrium of Mg(II) with ATP are

complicated. The intermediate exchange rate causes broad signals,

and this broadening results in unresolved Pfi signals which are

uninterpretable. To overcome this problem, the Sc(111)-ATP complex

possessing a slow exchange rate was selected.

In Chapter V, we indicated that M(III) forms 1:2 complexes with

ATP. However, 1:1 stoichiometry of M(111)-ATP complexes have been

assumed in the enzyme kinetic experiments designed to study the dissociation constants of these metal»nucleotide complexes (Morrison

& Cleland, 1983; Morrison & Cleland, 1980; Viola et a l., 1980). The calculated kinetic constants of M( 111) • ATP based on 1:1 stoichi oinetery would be erroneous if AK binds a 1:2 M( 1 1 1 )(ATP>2 complex. In addition to observing the nonproductive binding, the utilization of

Sc(111)ATP complex may also resolve the question of binding stoichiometry. 146

Sc : All*: AK

: 1.1 : I

15 : 1.1 : i.«

c : l.o : 1.1 l^r*W -V k^^M 1 *• W v V a ^ M 15

0 5 . i.i : 1.1

15 : I I : I.i Y,*Wi*-*''Wl V i,^

IS : I.i : i.i

IS : I.i : l «

- r~ (I - 1 0 - 2 0 |> p in.

Figure 7.1 31P NMR (202.5 MHz) spectra of Sc(ATP ) 2 (A, 1.6 mM ScClg,

3.14 mM ATP) titrated with a solution of 4.2 mM AK (B-G ). Spectral parameters: spectral width, 10,000 Hz; 60° pulse, no. of scans,

1000; LB, 3 Hz. 147

Table 7.1 31P NMR Parameters of Free and AK-Bound ATP

at 1 0 °C, pH 7.8

Complexes 6 (ppm) J (Hz)

a B Y JaB J By

ATP -10.7 -21.5 - 6 . 0 19.6 19.9

AK*ATP -10.7 -21.4 - 6 . 0 18.2 21.5

Sc(ATP) 2 - 1 2 . 0 - 2 0 . 1 -9.0 16.8 18.5

AK*Sc*ATPa - 1 2 . 1 -20.3 -9.0

Cd.ATP -10.4 -19.0 -4.4 18.0 15.8

Mg*ATP -10.3 -19.0 -5.5 15.8 15.8

AK*Mg*ATPb - 1 0 . 8 C -15.8 - 6 . 1c

aObtained from Figure 7.1.

^Obtained from Figure 7.4. cAn average of AK*Mg*ATP, MgATP and AK*ATP. 148

Figure 7.1, A shows the NMR spectrum of Sc (111) (ATP)^. When

AK was added stepwise, as shown in Figure 7.1, B-E, a new set of signals Pa' P^', and Py' formed. The intensities of these peaks increase gradually and remain constant after Sc:ATP:AK = 0.5:1.0:1.0.

The new signals P„', P^, and Py' could arise from four possible outcomes of binding, (a) They could represent free ATP in solution, that is released due to binding of AK to the 1:1 Sc(111)-ATP complex or the EDTA remaining in AK stock solution. This possibility can be ruled out since the coupling constants, Jag and J ^ , are different from those of free ATP. (b) The signals Pa'*, P^', and P ' may arise from AK»Sc(ATP)2. If true, one would predict a 1:2 stoichiometry for

AK:ATP, and this is inconsistent with the approximately linear increase of a', ft', and y' up to [AK]/[ATP] = 1.0. Further, the two

ATP molecules are likely to become nonequivalent in the AK»Sc(ATP ) 2 complex, yet only one set of new signals was observed, (c) It is also unlikely that Pa ' , P^'t and P ' represent AK«Sc»ATP since this would require the release of one equivalent of free ATP. (d) The only remaining possibility is that Pa% P^', and P ' represent the binary complex, AK»ATP, that is supported by the observed chemical shifts and coupling constants. This implies that ATP has a higher affinity than Sc(III)ATP to AK.

After the equilibrium has been reached, the signals Pa, Pg, and

PY in Figure 7.1, E and F, could be assigned to (a) free Sc(ATP)2,

(b) AK*Sc(ATP)2, or (c) AK*ATP»Sc. The fact that the ratio [a ', ft' ,

Y '] / [ a , B, y] does not further change when [AK]/[ATP]tota^ > 1 suggests that both species are enzyme bound, and reach an 149

Sc:AM’:AK

- 6 : < : I

l.! : 4 - I B

U : < : I

2.6 : 6 : 1 E

3.6 : 1 : 1

-ti -20 |i p 111

Figure 7.2 ^lp nmr spectra of a mixture of Cy-I^JATP'AK with the ratio of 4:1 (1.5 mM AK, 5.9 mM ATP) and titrated with SCCI 3 . The spectral parameters were the same as in Figure 6.1. The intensity variation of P^ at - 6 . 0 ppm were due to the * 7 0 isotope. 150 equilibrium. Thus, (a) can be ruled out. Between the other two possibilities, a, p, and y are tentatively assigned to AK»ATP»Sc since AK»Sc(ATP) 2 would possibly give two sets of signals, and each set represents 25% of the total ATP. However, a small portion of

Sc(ATP) 2 free in solution merging into this signal can not be ruled out.

In summary, the equilibrium in Figure 7.1 can be represented by the equation below:

. Sc( III )(ATP) 2 + 2AK ^ = 5= AK-ATP + AK*Sc (111)-ATP (eq. 7.1)

We examined the competition of ATP and Sc(III)«ATP for AK further by titrating the AK«ATP binary complex with Sc (111). This is shown in

Figure 7.2.

Figures 7.2, A-F, show the titration experiments of a 4:1 mixture of [y- ^ o ^ATP jAK with Sc (111). As the ratio of Sc (111) /

[y-^OjlATP/AK increases from 0/4/1 to 3.4/4/1, the intensities of

Pa', Pp', and P ' increase. From the coupling constants in Figure

7.2, and the equilibrium shown in Figure 7.1, it is obvious that the peaks of Pa, Pg, and Py are the average of E»ATP and free ATP.

However, Pa', Pg', and Py' may be due to an average of the AK»Sc»ATP and Sc(ATP ) 2 peaks. In the titration experiment of ATP with Sc(111), only 0.5 eq. of Sc( 111) (i.e. [Sc(III)]/[ATP] = 0.5 in Figure 5.1) is needed to convert Pa, Pg, Py to Pa', Pg', and Py'. The equilibrium 151

is represented by:

Sc(111) + 2ATP ==== Sc(III)(ATP) 2 (eq. 7.2)

However, in the presence of enzyme, a ratio of [Sc(III)]/[ATP] = 0.85 is needed to convert a, B, y, to a ', &', and y". The results of Figure 7.2 suggest that AK-ATP Is in equilibrium with AK»Sc»ATP and only in high concentrations of Sc(lII), can AK*ATP be converted to AK>Sc*ATP and

Sc(III)(ATP)2.

The experiments shown in Figure 7.1 and 7.2 may be summarized by

Scheme 7.1, assuming that the AK*M*ATP complex has a 1:1:1 stoichiometry.

E ATP .k . E.ATP

t Ki

k 2 ^ M k 4 ^M

Kg

H T n r . n*M1M. r ■ ------r f M* A TD1 r

E

Scheme 7.1

where Kj, K2, Kg, and K4 are defined as:

[AK*ATP] [M-ATP] Ko = [a Tp][A kI LmjLatpj

[AK* M-ATP] [AK*M-ATP] K. = Ko = [AK][M*ATP] [AK*ATP][Mj

and Kj K4 = KgKg 152

If the equilibrium process in Figure 7.1, E and F, is limited to

AK»ATP + Sc ==== AK«Sc»ATP, the association constant (K4 ) calculated

from the initial concentration and the ratio of species

[AK»ATP]/[AK*Sc«ATP] is at most 2xl03 M”1. This value could be even lower if the species of Pa, Pg, and P are a mixture of AK*Sc»ATP and

Sc(III)(ATP)2 - The K2 of ScATP was estimated as 10 6 ~ 107 M

(Morrison & Cleland, 1980; 1983), whereas the K^ of AK*ATP was

reported as 3 x 10^ M" 1 (Price et al., 1973). Thus, the ratio of

K2 /K4 and Kj/Kj is roughly equal to 10 3 ~ 104. This imply that the affinity of ATP for AK is much stronger than that of M»ATP.

The weaker interaction of Sc(III)-ATP with the enzyme can be

rationalized as follows. If it binds at the AK active site predominantly as a , 8 , y-tridentate and if the binding is mainly due to ionic interactions, this species probably possesses only one negative charge. Compared to the natural substrate MgATP (net charge, -2) and the nonproductive substrate ATP (net charge, -3), the low affinity is expected.

Isotope scrambling was also observed in Figure 7.2. The increasing HPY/HPa and decreasing HPg/HPa (HPa , HPg and HPy denote the peak intensities of Pa, Pg and Py in Experiments 7.2, A-D) indicate that the *70 originally labled at Py was transferred to P 0 through an enzyme catalyzed interconversion. The ATP is possibly activated by the tri valent Sc (111) or any remaining metal ion impurities.

In order to further verify this, an 31P NMR experiment of [y-

^OjlATP with incubating of AK was performed. As shown in Figure 153

[v -U021ATP:AK

1.0 : o

1.0 : 1.2

1.0 : 0 .<

1.0 : 0 .8

1.0 : 0 .0

1.0 : 1.0

1.0 : 1.2

0 - 1 0 -20 ppm

Figure 7.3 3*P NMR spectra of [y- 1 7 0 3]ATP (1.8 mM, pH 8.0)(A), and titrate d with varying concentrations of AK(B ~ G). 154

7.3, the presence of chelex-100, cation chelator could not prevent the incresase of HPY/HPa (i.e. the isotope scrambling. In the similar titration experiment of [a-l^jA T P with AK, ^ 0 isotope scrambling has not been observed (in Figure 6.1, the peak intensities do not change during the titration course). Therefore, the varying HPY/HPa and HP/HPa in Figure 7.3 are due to the isotope scrambling but not the binding of AK. At this stage, it seems that the impurity divalent metal ions instead of Sc(III) are the activator of the

interconverting reaction.

7.2 NMR Experiment of AK«Cd(II)«ATP

One can argue that the weak interaction of Sc(111) -ATP in the previous section is due to the trivalent, tridentate M(III). It

seems that a divalent M(II) is needed to address the question further. Cd(II) has been widely used in the approach of screw sense

specificity for AK. Table 7.2 lists the kinetic constants, 1^ and

Vmax* of Cd(II) complexed to various nucleotides that serve as substrates of AK from different sources (Kalbitzer et a l., 1983;

Tomasselli & Noda, 1983; Tomasselli et a l., 1984). The Km in all cases are on the order of 10"^ ~ 10“^ M and were reported as AK*CdATP complex. Considering the fact that the stability constant of CdATP is smaller than that of MgATP (Pecoraro et a l., 1984), the nonproductive binding of E»ATP will be more obvious.

Figure 7.4, A, represents a binary complex of AK«ATP with a ratio of 1.2:1.0. The parameters are given in Table 7.1, and are consistent with those in Figure 6.1. Figure 7.4, B-D, were obtained 155

Table 7.2 Kinetic Constants for the Interaction of Adenylate Kinase

from Beef Heart (AK1), Pig Muscle (AK2) and Yeast (AK3)

Substrate Vma)( X (gmol mg ' 1 m ln'1) •<„, (mM)

AKj AKZ AK3 AKj AK2 AK3

MgATP 1(10(536) 100(1500) 100(714) 0.37 0.06 0.1

CdATP 25 15 25.6 0.46 0.4 0.2

( Sp)MgATP(as) 9.3 1.4 64.0 0.63 0.2

(Rp)MgATP(as) 2.5 0.4 7.2 0.78 0.11

(Sp)MgATP(Bs) 0.15 0.005 0.2 0,92 0.17 0.50

(Rp)MgAIP(Bs) 0.01 0.002 0.02 1.53 0.12 0.73

( Sp)CdATP(as) 6.8 3.5 17.4 0.70 0.16

(Rp)CdAIP(ns) 0.3 (1.05 1.8 0.88 0.15

(Sp)CdAlP(us) 0.07 O.IJOB 0.025 1.35 0.15 0.62

(Rp)CdATP(Bs) 0.23 0.085 0.7 0.97 0.15 0.45

This table was obtained from (Kalbitzer et al., 19113; Tomasselli et al., 1979, Tomasselli et

al., 1984). 156

AKJATlMCdlll)

12:11 : i

1.2 : i.o : 0.5

1.2 : 1.0 : 1.0

1.2 : 1.0 : 2.0

i t 0 -10 20 p p hi

Figure 7.4 31P NMR‘spectra of the AK-ATP binary complex (A, 2.0 mM

AK, 1.8 mM ATP) titrate d with varying concentrations of CdCl2- 157

O 1 after various amounts of Cd(II) had been added. The P NMR parameters in these three spectra are the same as those in the AK*ATP binary complex. Higher ratios of Cd(II) were also attempted, however, sample precipitation occurred.

Since the NMR properties of Cd(II)-ATP are quite different from those of ATP and AK«ATP (Table 6.1), the signals in Figure 7.4 cannot be due to AK-Cd(II)-ATP or Cd(II)*ATP complex. A possible interpretation is that the addition of Cd(II) does not disturb the binary complex of AK-ATP appreciably. This would imply that ATP has higher affinitiy than Cd(II)*ATP for AK.

7.3 31P NMR Experiment of AK»Mg(II).ATP

One can criticize that the weak binding of Sc(111)ATP and

Cd(II)ATP to AK is due to the "wrong" structures of these two complexes. If this is true, MgATP can still bind tightly to the active site of AK. However, if the weak binding observed in the previous two sections is due to the stronger nonproductive binding of

ATP (i.e. ATP may bind stronger on AK than M«ATP does), the binding equilibrium of MgATP to kinases cannot simply be represented by

E + MgATP =*=* E* Mg ATP 158

Instead, an equilibrium of h*ATP, E*MgATP and MgATP may exist. This is illustrated by Scheme 7.1. It is this controversy that prompted me to perform the 31P NMR titration experiments of the AK«ATP binary complex with Mg(II).

Figure 7.5, A, shows the 3*P NMR spectrum of the AK»ATP binary complex. The chemical shifts and coupling constants, Jag and J ^ , correspond to the observations of AK-ATP made in Section 6.1. When the ratio of AK:Mg(II):ATP increases to 1:1:1, as shown in Figure

6.3. B, a broad resonance is observed. This resembles the resonances observed by Nageswara Rao et al. (1978., see Figure 1.1, B and C).

In their report, the broad PR signal was assigned as an average of

AK«ATP and AK*Mg»ATP. However, this possibility has been excluded in

Chapter VI. Thus, it is more reasonable due to an average of AK*ATP and Mg«ATP. We increased the ratio of AK:Mg(II):ATP to 1:10:1 by titrating Mg(II) into the sample shown in 7.5, B, and the result is illustrated in Figure 7.5, C. The Pp signal of ATP splits into three separate resonances. The tentative assignment for these signals:

(1) -15.8 ppm corresponds to AK»Mg»ATP, (2) -18.7 ppm is Mg»ATP, and

(3) -21.6 ppm represents AK*ATP. If this interpretation is correct, then the relative intensities of Mg*ATP and AK*ATP will decrease if more Mg(II) is added. As shown in Figure 7.5, D, when an additional

5 eq. of Mg(II) was added, the resonance at -15.8 ppm increased its relative peak intensity.

At this stage, it appears that the AK*Mg(II)»ATP system follows the equilibrium pictured in Scheme 7.1. The concentrations of the present complexes (i.e. Mg*ATP, AK»My*ATP, and AK»ATP) are dependent 159

i Y j \ ] i-° 0 V* ‘r^Vf.LVA'Y^.ri'i/ '’VvWMy^V I

^vA^,.,y'*Wv^A'fyV, \W

i i.i i.i ii.i y y V ' V H1 V * ’v- '

1.1 1.0 15.0 \ k » v

0 - 1 0 - 2 0 ppm

Figure 7.5 3*P NMR spectra of AK»ATP binary complex (A, 1.65 mM AK,

1.5 mM ATP) titrated with varying concentrations of Mg(II). Spectral parameters are the same as in Figure 6.1, except 4 Hz line on the broadening; 4,000 transitions for A,B, 10 Hz linebroadening; 10,000 transitions for C.D. 160 on the equilibrium constants (K^ ~ K^) as well as the amount of

Mg(II), ATP, and AK. The affinity of Mg*ATP for AK is very likely to be quite weak compared to that of ATP. It is, therefore, only under high concentrations of Mg(11) that the ternary complex AK»Mg«ATP will be observed.

Unfortunately, the possibility that the new peak resulted from the binding of S'-AdoP^ cannot be excluded. Although, no resonance for either free or Mg(II)-chelated 5'-MoP^ appear at -16 ppm (Figure

6.7), the chance for a chemical shift change is there if this compound is present as an enzyme bound form. 161

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The nonlinear least square fitting results

169 170

p„ of Ca(lI)AUP

OHO OF DATA POINTS IS 12 NO OT PARAMETERS IS I NO 0T INDEPENDENT VARIABLES IS I 0DELTA= .100000000-04 E= .500000000-04 FF= .400000000*01 GAHCR= .450000000*02 T= .200000000*01 TAU- .100000000-02 2ETA= .100000000-30 AL= .100000000*00 o fo rc e orr

0N0 or ITERATIONS = 20 OPHI = . 1003BS01D*03 LAMBDA * .100000000-00 OPARANETERS .986820050*00 OGAHHA = .853773660-06 LENGTH OF DB = .149781770*00 ODB CORRECTION VECTOR .132066560-02

OPTP INVERSE

OCOLUHNS I THROUGH I .313422930-03

OPARAHEIER CORRELATION COEFFICIENTS

OCOLUHNS 1 THR0U6H 1 I . 0000

I OBSERVED PREDICTED RESIDUAL

1 .101000000*02 .659304450*01 .455617590*01 .203686860*01 2 .988000000*01 .659304450*01 .616571190*01 .427332670*00 3 .966000000*01 .659304450*01 .799161810*01 -.139857360*01 4 .959000000*01 .659304450*01 .894978770*01 -.235674320*01 5 .947000000*01 .659304450*01 .922794940*01 -.263490480*01 6 .946000000*01 .659304450*01 .936218070*01 -.276913620*01 7 .934000000*01 .659304450*01 .939459270*01 -.280154810*01 B .933000000*01 .659304450*01 .956397200*01 -.297092750*01 9 .929000000*01 .659304450*01 .928750440*01 -.269445980*01 10 .928000000*01 .659304450*01 .969253030*01 -.309948570*01 It .927000000*01 .659304450*01 .100408030*02 -.344775830*01 12 .926000000*01 .659304450*01 .119319050*02 -.533BB607D*01

SID ONE - PARAMETER SUrFORT PLANE PARA ERROR LONER UrPER LONER UPPER 1 .5348I4B7D-01 .879865080*00 .109379100*01 .879865080*00 .10937910

Final value

I * I BID = .9868 pg of Ca(11)ADP 171

0N0 OF DATA POINTS 18 12 NO OF PARAMETERS IS I NO OF INDEPENDENT VARIABLES IS 1 ODELTA3 .100000000-04 E3 .500000000-04 TF= .400000000*01 6AHCR3 .450000000*02 T= .200000000*01 TAU= .100000000-02 ZETA3 .100000000-30 AL= .100000000*00 OFORCE OFF

ONO OF ITERATIONS 3 20 OPHI 3 .066790120*02 LAMBDA 3 .100000000-08 OPARAHETERS .905058240*00 06AHHA 3 .000000000*00 LENGTH OF OB 3 .695350600-01 OOB CORRECTION VECTOR .605282440-03

OPTP INVERSE

OCOLUHNS I THROUGH I .300186420-03

OPARAHETER CORRELATION COEFFICIENTS

OCOLUHNS 1 THROUGH I 1 .0 0 0 0

X OBSERVED PREDICTED RESIDUAL

1 .565000000*01 .659304450*01 .453493220*01 .205811230*01 2 .548000000*01 .659304450*01 .643914260*01 .153901920*00 3 .529000000*01 .659304450*01 .816688570*01 -.157384120*01 4 ,523000000*01 .659304450*01 .903091150*01 -.243786700*01 5 .514000000*01 .659304450*01 .878066420*01 -.219561970*01 6 .513000000*01 .659304450*01 .899024040*01 -.239719590*01 7 .501000000*01 .659304450*01 .100862660*02 -.349322150*01 e .500000000*01 .659304450*01 .104243060*02 -,3B3I2615D*01 9 .498000000*01 .659304450*01 .815279690*01 -.155975240*01 10 .497500000*01 .659304450*01 .987477290*01 -.320172840*01 11 .497000000*01 .659304450*01 .991163010*01 -.331858560*01 12 .496500000*01 .659304450*01 .101052080*02 -.351216370*01

STD ONE - PARAHETER SUPPORT PARA ERROR LONER UPPER LONER UFFER 1 .486358100-01 .887786620*00 .108232990*01 .887786620*00 .108232990*01

Final value

I 3 I BCD 3 .9851 172 Pa of Sr(II)ATP

Ai\>TYPE ATPH5RA.DAT I

ONO Or DATA POINTS IS 9 NO OF PARAMETERS IS I NO OF INDEPENDENT VARIABLES IS I ODELTA3 .100000000-04 E= .500000000-04 FF3 .400000000*01 6AHCR- .450000000*02 T3 .200000000*01 TAU= .100000000-02 ZETA3 .100000000-30 AL3 .100000000*00

0C0NVER6ENCE BY 6ANNA EPSILON TEST OCORRECTION VECTOR FOR LAST ITERATION HAS NOT USED

ONO OF ITERATIONS 3 2 OPHI 3 .366071620*02 LAMBDA 3 .100000000-02 OPARAHETERS . 99B5232ID*00 06AMMA 3 .000000000*00 LENGTH OF OB 3 .175041410-02 ODB CORRECTION VECTOR .150592620-04

OPTP INVERSE

OCOLUHNS 1 THROUGH 1 .296064130-03

OPARAMETER CORRELATION COEFFICIENTS

OCOLUHNS I THROUGH I 1 .0 0 0 0

X OBSERVED PREDICTED RESIDUAL

1 .551000000*01 .829404960*01 .458957170*01 .370447790*01 2 .520000000*01 .B2940496D*01 .630167820*01 .199237140*01 3 .495000000*01 .829404960*01 .698249180*01 .131155780*01 4 .484000000*01 .829404960*01 .740393240*01 .890117290*00 5 .475000000*01 .829404960*01 .801615810*01 .277831560*00 6 .46BOOOOOD*Oi .829404960*01 .919237790*0! -.898328250*00 7 .457000000*01 .829404960*01 .985448370*01 -.156043410*01 8 .442000000*01 .829404960*01 .108183530*02 -.252430340*01 9 .438000000*01 .829404960*01 .108845490*02 -.253043380*01

STD ONE - PARAMETER SUFPORT PLANE PARA ERROR LOHER UFFER LOHER UFFER 1 .368070430-01 .924909130*00 .107213730*01 .924909130*00 .10721373

Final value

I 3 I BID 3 .99B5 173

PB of Sr(II)ATP

ONO Or DATA POINTS IS 10 NO or PARAMETERS IS I NO OT INDEPENDENT VARIABLES IS I ODELTA= .100000000-04 E= .50000000D-04 FF= .400000000*01 6AMCR= .450000000*02 T= .200000000*01 TAU= .100000000-02 ZETA= .100000000-30 AL= .100000000*00

OCONVERGENCE BY EPSILON TEST

ONO OF ITERATIONS = 12 OPHI = .395610690*02 LANBDA = .100000000-08 OPARANETERS .102365890*01 OGAHNA = .000000000*00 LENGTH Or 08 = .155358160-02 ODB CORRECTION VECTOR .405360860-04

OPTP INVERSE

OCOLUHNS 1 THROUGH I .682286890-03

OPARAHETER CORRELATION COEFFICIENTS

OCOLUHNS I THROUGH I 1 .0 0 0 0

X ODSERVED PREDICTED RESIDUAL

1 .211300000*02 .829404960*01 .470888990*01 .358515980*01 2 .206800000*02 .829404960*01 .735952770*01 .934521940*00 3 .201500000*02 .829404960*01 .894253020*01 -.648480600*00 4 .199800000*02 .829404960*01 .858598700*01 -.291937400*00 5 .198300000*02 .829404960*01 .830223300*01 -.818938460-02 6 .196700000*02 .829404960*01 .838973300*01 -.956834000-01 7 .194200000*02 .829404960*01 .855740170*01 -.263352010*00 B .192500000*02 .829404960*01 .923831690*01 -.944267220*00 9 .192200000*02 .829404960*01 .944843430*01 -.115438470*01 10 .191700000*02 .829404960*01 .130925690*02 -.479B5I970*0I

STO ONE - PARAMETER SUPPORT PLANE PARA ERROR LOHER UFFER LOHER UFFER 1 .547641400-01 .914130590*00 .113318710*01 .914130590*00 .113318710*01

Final value

I = 1 B(I) = 1.0237 174

P a of Zn(11)AUP

ONO or DATA POINTS IS II NO OF PARAMETERS IS I NO Or INDEPENDENT VARIABLES IS I 0DELTA= .100000000-04 E= .500000000-04 rr= ,400000000»0l GANCR= .450000000*02 T= .200000000*01 TAU= .100000000-02 ZETA= .100000000-30 AL= .100000000*00

OCONVER6ENCE BY EPSILON TESY

ONO OP ITERATIONS = 16 OPHI = .126435240*03 LAHBDA * .100000000-00 OPARANETERS .103609060*01 OGAHHA s .000000000*00 LENGTH OF OB * .122290160-02 OOB CORRECTION VECTOR .398513600-04

OPTP INVERSE

OCOLUHNS I THROUGH 1 .106174860-02

OFARANETER CORRELATION COEFFICIENTS

OCOLUHNS 1 THROUGH 1 1 .0 0 0 0

I OBSERVED FREDICTED RESIDUAL

1 .101000000*02 .990340750*01 .503749830*01 .406593930*01 2 .960000000*01 .990340750*01 .642622600*01 .347726000*01 3 .953000000*01 .930349750*01 .684358300*01 .305303770*01 4 .939000000*01 .990348750*01 .732004180*01 .25B2615B0*0I 5 .927000000*01 .990348750*01 .703637670*01 .200651080*01 6 .916000000*01 .990340750*01 .068977120*01 .121371630*01 7 .912000000*01 .990340750*01 .951036900*01 .385119580*00 B .906000000*01 .990340750*01 .110666300*02 -.116314230*01 9 .902300000*01 ."90140750*01 .137632130*02 -.385972540*01 10 .902100000*01 .990340750*01 .137389230*02 -,3B3543520*01 11 .902000000*01 .390348750*01 .160728600*02 -.616937200*01

STD ONE - PARANEIER SUPPORT PARA ERROR LONER UFPER LONER urrER 1 .115063040*00 .864364480*00 .132781660*01 .064364480*00 .132701660*01

Final valut

1 = I BID = 1.0961 175 Pa Of Lu(III)ATP

At\>IVPE LUATPA.DAT I

ONO OF DATA POINTS IS 10 NO Or PARAMETERS IS 1 NO Or INDEPENDENT VARIABLES IS 1 ODELTA* . 10000000D-04 E= .500000000-04 PF= .100000000*01 6AKCR= .150000000*02 T- .200000000*01 TAU= .100000000-02 ZETA= .100000000-30 AL= .100000000*00

0C0NVER6ENCE BY EPSILON TEST

ONO OF ITERATIONS = 6 OPHI = .231522530*03 LAMBDA = .100000000-06 OPARANETERS .227813810*01 06AHNA = .000000000*00 LENGTH OP OB = .812042660-03 008 CORRECTION VECTOR .372005930-04

OPIP INVERSE

OCOLUHNS I THROUGH I .209855940-02

OPARAHETER CORRELATION COEFFICIENTS

OCOLUHNS I THROUGH I 1 .0 0 0 0

X OBSERVED FREDICTED RESIDUAL

1 .105999000*02 .181206810*02 .104935380*02 .792711290*01 2 .105000000*02 .184206810*02 .127916500*02 .562903030*01 3 .104000000*02 .184206810*02 .140063420*02 .441433860*01 4 .103800000*02 .184206810*02 .150087670*02 .311191340*01 5 .103600000*02 .184206810*02 .152416420*02 .317903910*01 6 .103300000*02 .184206810*02 .159110570*02 .250962380*01 7 .102600000*02 .184206810*02 .241326300*02 -.571191910*01 e .102599000*02 .184206810*02 .233065470*02 -.488586590*01 9 .102598000*02 .184206810*02 .226315970*02 -.421091580*01 10 .102597000*02 .184206810*02 .223238920*02 -.390321090*01

STO ONE - PARAHETER SUPFORT PARA ERROR LOHER UPPER LOHER INTER 1 .232346430*00 .181380520*01 .274319100*01 .181380520*01 .274319100*01

Pinal value

I = I B(I) = 2.2785 I

176

P0 of Lu(III)ATP

ONO or DATA POINTS IS 11 NO OF PARAMETERS IS 1 NO OT INDEPENDENT VARIABLES IS I ODELTA= .IOOOOOOOD-04 E= .500000000-04 Fr= .400000000*01 6ANCR= .450000000*02 T= .200000000*01 TAU= .100000000-02 ZETA= .100000000-30 AL= .100000000*00

((CONVERGENCE BY EPSILON TEST

OND OF ITERATIONS = 4 OPHI = .416540200*03 LANBOA = .100000000-04 OPARAHETERS .100012870*01 OGANHA = .000000000*00 LENGTH OF OB = .414501920-02 ODB CORRECTION VECTOR -.690199470-04

OPTP INVERSE

OCOLUMNS 1 THROUGH I .278342880-03

OPARANETER CORRELATION COEFFICIENTS

OCOLUHNS 1 THROUGH I 1 .0 0 0 0

0 I OBSERVED PREDICTED RESIDUAL

1 .212999000*02 .184206810*02 .869613150*01 .972454920*01 2 .208000000*02 .184206810*02 . 1032B500D*02 .809218040*01 3 .201000000*02 .184206810*02 .124458440*02 .597483630*01 4 .196500000*02 .184206810*02 .131589600*02 .526172060*01 5 .194000000*02 .184206810*02 .123239930*02 .609668770*01 6 .191000000*02 •IB4206BID*02 .12B5B6670*02 .556201410*01 7 .189000000*02 .184206810*02 .139880470*02 .443263370*01 B .181000000*02 .184206810*02 .190213800*02 -.600699390*00 9 .180000000*02 .184206810*02 .185280000*02 -.107319350*00 10 .179600000*02 .1B42068ID*02 .203194710*02 -.189879070*01 11 .179500000*02 .184206810*02 .284895460*02 -.100688660*02

0 STD ONE - PARANETER SUPPORT PARA ERROR LOHER UFPER LOHER UFFER 1 .107676950*00 .167297480*01 .210368268*01 .167297480*01 .210368260*01

Final value

I = I B(l) = 1.0883 177 PY o f LU(III)ATP

ONO o r DATA POINTS IS 10 NO OF PARAMETERS IS I NO OF INDEPENDENT VARIABLES IS 1 ODELTA3 .100000000-04 E= .500000000-04 rr= .400000000*01 6AHCR= .450000000*02 T= .200000000*01 TAU= .100000000-02 ZETA= .100000000-30 AL= .100000000*00

OCONVERGENCE BY EPSILON TEST

ONO OF ITERATIONS = 3 OPHI = .410691490*03 LAMBDA = .100000000-03 OPARAHETERS .190220400*01 06AMMA = .000000000*00 LENGTH OP OB = .357556520-01 ODB CORRECTION VECTOR -.393475520-04

OPTP INVERSE

OCOLUHNS I THROUGH 1 .129789900-05

OPARAMETER CORRELATION COErriCIENTS

OCOLUHNS I THROUGH 1 1 .0 0 0 0

X OBSERVED PREDICTED RESIDUAL

1 .559990000*01 .184206810*02 .875927300*01 .966140770*01 2 .565000000*01 ,184206BID*02 .487770040*01 .135429800*02 3 .570000000*01 .184206810*02 .132164390*02 .520424200*01 4 .575000000*01 .184206810*02 .123352670*02 .609541370*01 5 .578000000*01 .184206810*02 .182609910*02 .159689870*00 6 .580000000*01 .184206810*02 .149010010*02 .351960020*01 7 .584000000*01 .184206810*02 .155764570*02 .284422410*01 B .586000000*01 .184206810*02 .166745560*02 .174612470*01 9 .587000000*01 .184206810*02 .231205930*02 -.469991250*01 10 .587011000*01 .1B4206B1D*02 .240933480*02 -.567266700*01

STD ONE - PARAHETER SUPPORT PARA ERROR LOHER UPPER LOHER UFPER 1 .777045160-02 .188666390*01 .191774570*01 .188666390*01 .191774570*01

Final value

I = I B(I) = 1.9022 178 He O f Lu(III)ATP

Af\>TVPE LU8HNR.DAT I

ONO Or DATA POINTS IS 8 NO OF PARAMETERS IS 1 NO OT INDEPENDENT VARIABLES IS I ODELTA- . IQQQOOOQD-04 E= .500000000-04 FF= .400000000*01 GAHCR= .450000000*02 T= .200000000*01 TAU= .100000000-02 ZETA- .100000000-30 AL= .100000000*00

OGONVERGENCE BY EPSILON TEST

ONO OF ITERATIONS - I I OPHI = .114970780*03 LAMBDA = .100000000-08 OPARANETERS . 183792780*01 06ANNA = .000000000*00 LENGTH OF OB - . 44BB774BD-0I ODB CORRECTION VECTOR -.391533370-04

OPTP INVERSE

OCOLUHNS 1 THR0U6H I .829992500-08

OPARAMETER CORRELATION COEFFICIENTS

OCOLUHNS I THROUGH 1 1 .0 0 0 0

0 X OBSERVED PREDICTED RESIDUAL

1 .856000000*01 .169065530*02 .845639040*01 .845016260*01 2 .847000000*01 .169065530*02 .169119950*02 -.544167000-02 3 .842000000*01 .169065530*02 .129172530*02 .398929350*01 4 .838000000*01 .169065530*02 .130731890*02 .383336420*01 5 .835000000*01 .169065530*02 .138951100*02 .301144340*01 6 .832000000*01 .169065530*02 .177280880*02 -,B215349!D*00 7 .831900000*01 .169065530*02 .176818730*02 -.775320280*00 8 .831800000*01 .169065530*02 .185225750*02 -.161602190*01

0 STD ONE - PARAMETER SUFPORT PARA ERROR LOHER UPPER LOHER UPFER 1 .369217100-02 .183054330*01 .184531200*01 .183054330*01 ,184531200*01

Final value

I = B(I) = 1.8379 179 H2 of Lu(III)ATP

Al\>TYPE LU2HHR.DAT I

ONO OF DATA POINTS IS B NO OF PARAMETERS IS I NO Or INDEPENDENT VARIABLES IS 1 0DELTA= .100000000-04 E- .500000000-04 If* .400000000(01 GAHCR= .450000000(02 T» .200000000(01 TAU= .100000000-02 ZETA= .100000000-30 AL= .100000000(00

OCONVERGENGE BY EPSILON TEST

ONO OF ITERATIONS = I I OPHI = .190299570(03 LAMBDA c .100000000-08 OPARANETERS .175113910(01 06ANHA * .000000000(00 LENSTH OF OB = .2B55B3020-02 ODB CORRECTION VECTOR -.750270090-04

OPTP INVERSE

OCOLUHNS 1 THROUGH I .690930930-03

OPARAHETER CORRELATION COEFFICIENTS

OCOLUHNS I THR0U6H 1 1 .0 0 0 0

I OBSERVED PREDICTED RESIDUAL

1 .826700000(01 .169065530(02 .806340860(01 .884314420(01 2 .816500000(01 .169065530(02 .104710800(02 .643547250(01 3 .014500000(01 .169065530(02 .125233800(02 .438317310(01 4 .809900000(01 .169065530(02 .140103740(02 .289617070(01 5 .807000000(01 .169065530(02 .207664800(02 -.385992720(01 6 .806990000(01 .169065530(02 .189720190(02 -.206546630(01 7 .806960000(01 .169065530(02 .187617450(02 -.185519150(01 0 .806910000(01 .169065530(02 .214316040(02 -.452505070(01

STD ONE - PARAHEIER SUPPORT PLANE PARA ERROR LOHER u ite r LOHER UPPER 1 .137052580(00 .147703390(01 .202524420(01 .147703390(01 .202524420(01

Final valut

I * I 0(1) = 1.7511 180 Hj' of LuIIII)ATP

ONO OF DATA POINTS IS B NO OF PARAMETERS IS I NO OF INDEPENDENT VARIABLES IS I ODELTA* .100000000-04 E= .500000000-04 FF= .400000000*01 GANCR* .450000000*02 T= .200000000*01 TAU= .100000000-02 2ETA= .100000000-30 AL= .100000000*00

0C0NVER6ENCE BY BANNA EPSILON TEST OCORRECTION VECTOR FOR LAST ITERATION HAS NOT USED

ONO OF ITERATIONS = 10 OPHI = .249494420*03 LAHBDA = .100000000-00 OPARANETERS .189037500*01 OGANNA = .000000000*00 LENGTH OF OB * .191504450-02 OOB CORRECTION VECTOR -.5366038B0-04

OPIP INVERSE

OCOLUNNS 1 THROUGH 1 .314058000-02

OPARANETER CORRELATION COEFFICIENTS

OCOLUNNS I THROUGH I 1 .0 0 0 0

I OBSERVED PREDICTED RESIDUAL

I .614200000*01 .169065530*02 .869153980*01 .821201370*01 2 .610000000*01 .169065530*02 .975155710*01 .715199600*01 3 .607000000*01 .169065530*02 .103828110*02 .652374210*01 4 .602000000*01 .169065530*02 .125679610*02 .433059200*01 5 .597000000*01 .169065530*02 .215193620*02 -.461340860*01 6 .596990000*01 .169065530*02 .197433510*02 -.203679840*01 7 .596970000*01 .169065530*02 .194553450*02 -.254879160*01 B .596910000*01 .169065530*02 .227114060*02 -.5B0485260*01

STD ONE - PARANETER SUPPORT PARA ERROR LOHER UFPER LOHER 1 .334569480*00 .122123600*01 .255951400*01 .122123600*01 .255951400*01

Final value

I = 1 BI D = ' 1.8904 181 Jap of AK-ATP

OND or DATA POINTS IS 10 NO OT PARAMETERS IS I NO Or INDEPENDENT VARIABLES IS I ODELTA3 .100000000-04 E3 .500000000-04 FFS .400000000*01 GANCR3 .450000000*02 T= .200000000*01 TAU= .100000000-02 ZETA3 .10000000D-30 AL= .100000000*00

OCONVERGENCE BY EPSILON TEST

ONO OF ITERATIONS 3 20 OPHI 3 .161273550*03 LAMBDA » .100000000-08 OPARAHETERS .104220050*01 06AHHA 3 .000000000*00 LEN6TH OF OB 3 .126047450-02 ODB CORRECTION VECTOR -.509332220-04

OPTP INVERSE

OCOLUNNS I THR0U6H I .163274080-02

OPARANETER CORRELATION COEFFICIENTS

OCOLUHNS 1 THROUGH I I . 0000

X OBSERVED PREDICTED RESIDUAL

1 .197000000*02 .103089530*02 .647680050*01 .383215220*01 2 .196000000*02 .103089530*02 .702740710*01 .328154550*01 3 .193000000*02 .103089530*02 .844012760*01 .186882510*01 4 .189000000*02 .I030B953D*02 .491711610*01 .539183650*01 5 .187000000*02 .103089530*02 .525754700*01 .505140570*01 6 . !B300000D*02 .!030B953D*02 .660102280*01 .370792980*01 7 .182000000*02 .103089530*02 .125885710*02 -.227961840*01 8 .181900000*02 .103089530*02 .111978860*02 -.88B9329BD*00 9 .181500000*02 .103089530*02 .148104990*02 -.450154650*01 10 .181490000*02 ,1030B953D*02 .164521590*02 -.614320630*01

STD ONE - PARANETER SUPFORT PARA ERROR LOHER UFFER LOHER UFFER 1 .171048370*00 .700103760*00 .138429720*01 .700103760*00 .138429720*01

Final value

I 3 I B(I) 3 1.0422 JpY of AK-ATP

As\>TYPE LEU5Hl.DAT I

ONO OF DATA POINTS IS 10 NO OF PARAMETERS IS I NO Or INDEPENDENT VARIABLES IS 1 ODELTA= .100000000-04 E= .500000000-04 FF= .400000000+01 8AHCR= .450000000*02 T= .200000000*01 TAU- .100000000-02 ZETA= .100000000-30 AL= .100000000*00

OCONVERGENCE BY 6ANNA EPSILON TEST OCORREGTION VECTOR FOR LAST ITERATION HAS NOT USED

ONO Or ITERATIONS * 5 OPHI = .501072010*02 LANBDA = .100000000-05 OPARAMETERS .902478950*00 OGAMMA = .000000000*00 LENGTH OF OB = .216420920-02 ODB CORRECTION VECTOR .266070560-04

OPTP INVERSE

OCOLUHNS 1 THROUGH I .604501950-03

OPARAMETER CORRELATION COEFFICIENTS

OCOLUHNS 1 THROUGH 1 1 .0 0 0 0

X OBSERVED PREDICTED RESIDUAL

1 .200000000*02 .103089530*02 .610578860*01 .420316400*01 2 .203500000*02 .103089530*02 .010756110*01 .220139150*01 3 .206500000*02 .103089530*02 .886013740*01 .144881520*01 4 .200000000*02 .103089530*02 .114526110*02 -.114365030*01 5 .213000000*02 .103089530*02 .104205690*02 -.111616020*00 6 .213900000*02 .103089530*02 .123008610*02 - . I99I90BID*01 7 .214500000*02 .103089530*02 .121315670*02 -.182261410*01 8 .214600000*02 .103089530*02 .126182890*02 -.230933640*01 9 .214650000*02 .103089530*02 .135626020*02 -.325364970*01 10 .214660000*02 .103089530*02 .133033010*02 -.299434B0D*0I

STD ONE - PARAMETER SUPFORT PLANE PARA ERROR LOHER UFFER LOHER UPPER 1 .624771650-01 .857524620*00 .110743330*01 .857524620*00 .110743330*01

Final value

I 0(1) * .9825 183 APa of AK-ATP

ONO OF DATA POINTS IS I I NO OT PARAMETERS IS I NO OP INDEPENDENT VARIABLES IS I ODELTA3 .100000000-04 E3 .500000000-04 TF= .400000000*01 6ANCR3 .450000000*02 T3 .200000000*01 TAU= .100000000-02 ZETA3 .100000000-30 AL3 .100000000*00

OCONVERGENCE BY GANNA EPSILON TEST OCORRECTIDN VECTOR TOR LAST ITERATION HAS NOT USED

ONO Or ITERATIONS 3 4 OPHI 3 .762032970*02 LANBOA 3 .100000000-04 OPARAHETERS .100241520*01 OGANHA 3 .000000000*00 LENGTH OP OB 3 .153303570-04 008 CORRECTION VECTOR -.670230640-07

OPTP INVERSE

OCOLUNNS I THROUGH I .782109200-04

OPARANETER CORRELATION COEFFICIENTS

OCOLUNNS I THROUGH I 1 .0 0 0 0

X OBSERVED PREDICTED RESIDUAL

1 .360000000*01 .103001530*02 .853780110*01 .177115160*01 2 .300000000*01 .103009530*02 .733315120*01 .297580150*01 3 .410000000*01 .103003530*02 .807721720*01 .143173550*01 4 .460000000*01 .103089530*02 .913293990*01 .117601270*01 5 .515000000*01 .103089530*02 .100369000*02 .272052830*00 G .500000000*01 .103089530*02 .123441870*02 -.203523440*01 7 .628000000*01 .103083530*02 .149736730*02 -.466472050*01 e .629000000*01 .103089530*02 .12646BI70«02 -.233786400*01 9 .630000000*01 .103083530*02 .129171420*02 -.260818330*01 10 .630500000*01 .103089530*02 .132412590*02 -.293230660*01 II .631000000*01 .103089530*02 .140526880*02 -.374373570*01

STD ONE - PARAMETER SUPPORT PARA ERROR LOHER UPFER LOHER UPPER 1 .244257790-01 .953563660*00 .105126680*01 .953563660*00 . I05I266BD*0I

Final value

I 3 I B(I) 3 .1.0024 184

APy of AK-ATP

OND OF DATA POINTS IS 9 NO OT PARAMETERS IS I NO OF INDEPENDENT VARIABLES IS I ODELTA- .10000000D-04 E= .500000000-04 FF= .400000000*01 6ANCR= .450000000*02 T= .200000000*01 TAU= .100000000-02 IETA= .100000000-30 AL= .100000000*00

OCONVERSENCE BY 6ANNA EPSILON TEST 0C0RRECT10N VECTOR FOR LAST ITERATION NAS NOT USED

ONO OF ITERATIONS = I I OPHI = .419504320*02 LAMBDA « . lOOOOOOOD-OB OPARAMETERS .100730000*01 OSANMA = .000000000*00 LEN6TH OF OB = .666228330-02 Odb correction vector .445066790-04

OPTP INVERSE

OCOLUNNS I THROUGH 1 .178510670-03

OPARANETER CORRELATION COEFFICIENTS

OCOLUHNS 1 THROUGH 1 1.0000

0 X OBSERVED PREDICTED RESIDUAL

I .350000000*01 .103089530*02 .858010520*01 .172884740*01 2 .380000000*01 .1030B953D*02 .792006950*01 .238888310*01 3 .470000000*01 .103089530*02 .879531000*01 .151364260*01 4 .500000000*01 .103089530*02 .120373650*02 -.I72B4125D*01 5 .530000000*01 .103089530*02 .141930760*02 -.388412330*01 6 .531000000*01 .103089530*02 .123796010*02 -.207064800*01 7 .531100000*01 .103089530*02 .120586900*02 -.174973750*01 0 .532000000*01 .103089530*02 .125108830*02 -.220193000*01 9 .532700000*01 .103089530*02 .111409130*02 -.831960100*00

0 STD ONE - PARAMETER SUFPDRT PARA ERROR LOHER UPPER LOHER UFFER 1 .305953420-01 .946189320*00 .106857070*01 .946189320*00 .106057070*01

Final value

1 B(I) = ' 1.0074