BACTERIAL PHOSPHATIDYLINOSITOL-SPECIFIC PHOPHOLIPASE C:

INSIGHTS INTO ENZYMATIC MECHANISM THROUGH NMR, PROTEIN

ENGINEERING, AND LINEAR FREE ENERGY RELATIONSHIPS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy

in the Graduate School of the Ohio State University

By

Alexander V. Kravchuk, M.S.

*****

The Ohio State University

1999

Dissertation Committee: Approved by

Professor Ming-Daw Tsai, Adviser

Professor Lawrence Berliner

Professor Dehua Pei Adviser

Department of Chemistry IUVfl Number: 9951683

U l V d L l "

UMI Microform 9951683 Copyright 2000 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.

Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 ABSTRACT

Structure, function, and mechanism of phosphatidylinositol specific phospholipase C (PI-PLC) from Bacillus thuringiensis have been studied by multidimensional nuclear magnetic resonance (NMR) spectroscopy, protein engineering, and linear free energy relationships (LFER).

Determination of the pKaS of the histidine side chains in the wild type (WT) enzyme and two active site mutants has conclusively established a complex nature of the

PI-PLC’s pH-rate profile, where decrease of the activity at the extreme pHs cannot be explained by simple protonation of a general base or deprotonation of a general acid.

Most likely, pH-rate profiles of PI-PLC are governed by elaborate acid-base equilibria in the enzyme’s active site, effecting its extensive hydrogen bond network.

A single active site mutation, arginine-69 to aspartate, has created a metal binding site with sub-millimolar affinity for both magnesium and calcium ions. Metal ions have partially restored mutant’s activity, but even fully activated R69D falls far below the

WT’s level. Further examination of the created metal binding site has revealed that it is composed of aspartate-33, aspartate-67, aspartate-69, and glutamate-117. D33N/R69D double mutant favors magnesium over calcium ions, albeit the discrimination comes at the expense of slightly lower activity. Sigmoidal nature of the rate vs. metal ion concentration plots for the double mutant suggests that the second mutation has created a molecular switch in the active site, which is turned on by the addition of a metal ion.

Site-directed chemical modification has been used to elaborate function of arginine-69 in the enzyme catalysis. Consequences of the structure of the side chain at that position on enzyme’s activity, non-bridging thio-effects, and stereoselectivity have been systematically examined. The results have established that bi-dentate functional group in the place of Arg69 is essential for the enzyme’s activity. The proposal that arginine-69 interacts with both pro-S oxygen of the phosphate group and 2-OH of inositol

(incoming nucleophile in the reaction) seems to be the most consistent with the available functional and structural data.

LFER studies have examined effects of the pKa of the leaving group on the rates of both enzymatic and non-enzymatic reactions. Analysis of the Bronsted coefficients established that both enzymatic and imidazole-catalyzed reactions proceed through essentially identical, slightly dissociative, transition states. Additionally, it has been shown that replacement of a non-bridging phosphate oxygen by sulfur does not alter reactivity of the inositol phosphate diesters.

Ill ACKNOWLEDGMENTS

I would like to thank my adviser, Ming-Daw Tsai, for his support, both intellectual and financial, his encouragement and patience throughout of ups and downs of my graduate career.

Most of this work would be impossible without advice and intellectual input from professor Karol Bruzik from University of Illinois at Chicago. His lab has also supplied numerous substrate analogs used in this work. 1 have also learned a lot of synthetic organic chemistry during my ten week sabbatical in Prof. Bruzik's lab in summer of 1998 with the help and under the supervision by Robert Kubiak.

I am grateful to Dr. In Ja Beoyn and Dr. Charles Cottrell from OSU Campus

Chemical Instrument Center for teaching me high-field NMR of proteins and for their patience in solving my numerous problems and answering even more numerous questions related to my NMR experiments. Dr. Karl Vermillion provided an excellent technical support at the NMR laboratory of OSU Department of Chemistry.

I wish to thank Hua Liao for helpful discussions and valuable help with various aspects of the PI-PLC project. Li Zhao provided indispensable assistance in site-directed mutagenesis and protein purifications. 1 am indebted to Dr. Suzette Riddle for her help and instructions in the beginning of my graduate research.

IV VITA

February 3, 1967 ...... Bom - Slavuta, Ukraine

1989...... M.S., Moscow State University, Russia

1989 - 1993...... Researcher,

Institute of Biochemistry and Physiology of

Microorganisms, Russian Academy of Sciences,

Pushchino, Russia

1993 - present...... Graduate Teaching and Research Associate,

The Ohio State University

PUBLICATIONS

1. Hondal, R.J., Zhao, Z., Kravchuk. A V.. Liao, H., Riddle, S R., Bruzik, K.S. and Tsai, M.-D. (1999) “Mechanism of phosphatidylinosito 1-specific phospholipase C revealed by protein engineering and phosphorothioate analogs of phosphatidylinositol”. ACS Symp. Sen, 718, 109-120.

2. Hondal, R. J.; Zhao, Z.; Kravchuk. A. V.: Liao, H.; Riddle, S. R.; Yue, X.; Bruzik, K. S.; Tsai, M.-D. “Mechanism of phosphatidylinosito 1-specific phospholipase C: a unified view of the mechanism of catalysis”. Biochemistry (1998), 37(13), 4568-4580. 3. Hondal, R. J.; Zhao, Z.; Riddle, S. R.; Kravchuk. A. V : Liao, H.; Bruzik, K. S.; Tsai, M.-D. “Phosphatidylinositol-specific phospholipase C. 3. Elucidation of the catalytic mechanism and comparison with ribonuclease A.” J. Am. Chem. Soc. (1997), 119(41), 9933-9934.

4. Hondal, R. J.; Riddle, S. R.; Kravchuk. A. V.: Zhao, Z.; Liao, H.; Bruzik, K. S.; Tsai, M.-D. “Phosphatidylinositol phospholipase C: kinetic and stereochemical evidence for an interaction between arginine-69 and the phosphate group of phosphatidylinositol”. Biochemistry (1997), 36(22), 6633-6642.

5. Sakharovsky, V. G.; Kravchuk. A. V.: Buzilova, I. G.; Kozlovsky, A. G. “5- Lactone of 2,3-anhydromevalonic acid is a novel metabolite of the alkaloid- producing strain Pénicillium sizovae." Prikl. Biokhim. Mikrobiol. (1994), 30(6), 794-8.

6. Litvinenko, L. A.; Petrikevich, S. B.; Kravchuk. A. V.: Grishchenkov, V. G.; Boronin, A. M. “Catabolism of caprolactam and its intermediates by industrial strains Pseudomonas putida BS394 containing various CAP plasmids.” Mikrobiologiya (1993), 62(3), 447-52

7. Litvinenko, L. A.; Kravchuk. A. V.: Petrikevich, S. B.; Sacharovsky, V. G.; Ivanitskaya, Ju. G.; Gulamova, D. E. “Influence of heavy water on growth, glucose assimilation, and stability of Escherichia coli to freezing-thawing” Mikrobiologiya (1992), 61(6), 1030-7.

FIELDS OF STUDY

Major Field: Chemistry

VI TABLE OF CONTENTS

Page

Abstract...... ii

Acknowledgments ...... iv

Vita...... V

List of Tables...... xii

List of Figures ...... xiv

Abbreviations ...... xx

Chapters:

1. Introduction ...... 1

I. I Mammalian PI-PLCs...... 2

1.1.1 Structure...... 2

1.1.2 Function and ..regulation...... 4

1.1.3 Mechanism...... 8

1.2 Bacterial PI-PLC...... 10

1.2.1 Structure...... 10

1.2.2 Function and specificity ...... 12

1.2.3 Mechanism...... 12

VII 1.2.4 Comparison with RNase A ...... 15

NMR studies of bacterial PI-PLC ...... 16

2.1 Introduction ...... 16

2.1.1 NMR of large proteins ...... 16

2.1.2 pH-activity profiles of WT and mutant PI-PLC ...... 17

2.1.3 “Hydrophobic” activation of PI-PLC ...... 19

2.2 Materials and Methods ...... 21

2.2.1 Protein expression ...... 22

2.2.2 Protein purification ...... 24

2.2.3 Uniform and specific labeling of PI-PLC with '^C and '^N ...... 25

2.2.4 Sample preparation for NMR experiments ...... 26

2.2.5 1D and 2D NMR experiments ...... 26

2.2.6 Determinations of the pKaS of histidines in WT enzyme

and active site mutants...... 27

2.2.7 Protein-ligand interactions monitored by NMR ...... 28

2.3 Results and Discussion ...... 28

2.3.1 ID proton, 2D NOESY and HSQC NMR as monitors

of global protein structure ...... 28

2.3.2 Towards the total NMR assignment of PI-PLC ...... 37

2.3.3 pKaS of the histidine residues in WT PI-PLC

and selected active site mutants...... 41

vm 2.3.4 Binding of substrate analogs to PI-PLC

as monitored by HSQC ...... 47

3. Design and characterization of a catalytic metal binding site ...... 54

3.1 Introduction ...... 54

3.2 Materials and Methods ...... 56

3.2.1 Mutants...... 56

3.2.2 Radioactive activity assay of PI-PLC with [^H]-PI substrate ...... 57

3.2.3 R69D NMR titration by Ca'^ ...... 58

3.3 Results and Discussion ...... 58

3.3.1 Metal activation and metal specificity of R69D mutant ...... 59

3.3.2 Effect of calcium binding on R69D structure and dynamics ...... 63

3.3.3 Characterization of the metal binding site ...... 66

3.3.4 Low catalytic efficiency of the metal ions:

possible explanations, implications and new directions ...... 69

4. Catalytic role of arginine-69 in B. thuringiensis PI-PLC:

site-directed chemical modifications and thio-effects ...... 71

4.1 Introduction ...... 71

4.1.1 Phosphorothioates as mechanistic probes

of enzymatic reactions ...... 71

4.1.2 Thio-effects of RNase A and PI-PLC: lysine vs. arginine ...... 73

IX 4.1.3 Chemical modification of cysteine:

extension of the genetic code ...... 73

4.2 Materials and Methods ...... 76

4.2.1 Synthesis of dithio-bis(acetylamidine) ...... 76

4.2.2 Axg69—>Cys mutant ...... 77

4.2.3 Quantification of free thiol with DTNB ...... 78

4.2.4 Chemical modification reactions ...... 78

4.2.5 Native PAGE and isoelectro focusing ...... 79

4.2.6 Mass-spectrometry of modified proteins ...... 79

4.2.7 ^’P-NMR assays with DPPsI ...... 79

4.2.8 Determination of bridging thio-effects ...... 80

4.3 Results and Discussion ...... 81

4.3.1 Chemical modification of R69C mutant and

characterization of the modified proteins ...... 81

4.3.2 Effect of the side chain structure on activity and thio-effects 83

5. Physico-chemical characterization of intramolecular phosphoryl

transfer in inositolphosphodiesters ...... 93

5.1 Introduction ...... 93

5.1.1 Reactions of phosphate esters in chemistry and biology ...... 93

5.1.2 Linear free energy relationships: probes of the transition

states for chemical and enzymatic reactions ...... 95

5.2 Materials and Methods ...... 96

X 5.2.1 Synthesis of aryl phosphoinositides ...... 96

5.2.2 ^*P-NMR assays ...... 98

5.2.3 Purification of Rp phosphorothioates ...... 99

5.3 Results and Discussion ...... 100

5.3.1 Non-enzymatic reactions of inositol phosphodiesters ...... 103

5.3.2 LFER investigation of the enzymatic reaction ...... 108

Bibliography ...... 114

XI LIST OF TABLES

Table Page

2.1 pKa's o ftitratable histidines in WT PI-PLC and D—>N active site

mutants...... 45

3.1 Kinetic parameters of R69D PI-PLC with earth-alkaline metals ...... 59

4.1 Electrospray ionization mass-spectrometry analysis of PI-PLC R69C

mutant and its modifications ...... 82

4.2. Specific activities of PI-PLC with different side-chains at position 69

toward oxygen and phosphorothioate substrates ...... S3

4.3 Non-bridging thio-effects and stereoselectivity of PI-PLC with

different side-chains at position 69 ...... 85

5.1. Rate constants of imidazole-catalyzed intramolecular

transestérification of aryl inositol phosphodiesters ...... 106

XU 5.2. Maximal activities of WT PI-PLC in the intramolecular

transphosphorylation of aryl inositol phosphodiesters ...... 110

5.3. Summary of Bronsted coefficients for non-enzymatic (imidazole-

catalyzed) and enzymatic (WT PI-PLC) intramolecular

transphosphorylation of aryl inositol phosphodiester ...... 111

X l l I LIST OF FIGURES

Figure Page

1.1 Domain architecture of mammalian PI-PLCs ...... 3

1.2 Reactions catalyzed by PI-PLCs ...... 3

1.2 Crystal structures of the bacterial PI-PLC from Bacillus cereus (A)

and truncated (Al-132) mammalian PI-PLC 51 (B)...... 7

1.4 General acid/base mechanism proposed for PI-PLC ...... 8

1.5 Schematic representation of the active site of mammalian PI-PLC Ô1...... 9

1.6 Schematic representation of the active site of bacterial PI-PLC

from Bacillus cereus...... II

1.7 Current understanding of the active site residues’ participation

in the catalysis by bacterial PI-PLC...... 14

XIV 2.1 Two potential models for the activation of the bacterial PI-PLC

by hydrophobic part of its substrate ...... 20

2.2 One-dimensional proton NMR spectra of WT, H32A, D274A,

D274E, H82A, and D33A in D.Q at 600 MHz ...... 30

2.3 Two-dimensional phase-sensitive NOESY NMR spectra of WT,

H32A, D274A, D274E, H82A, and D33A in D.O at 600 MHz ...... 31

2.4 Backbone amide region of '^N-'H HSQC spectrum of the uniformly

'^N-labeled WT PI-PLC acquired at 600 MHz at 37°C ...... 33

2.5 Backbone amide region of '^N-'H HSQC spectrum of the uniformly

'^N-labeled WT PI-PLC acquired at 800 MHz at 37°C ...... 34

2.6 Backbone amide region of *^N-'H HSQC spectrum of the uniformly

'^N-labeled R69D PI-PLC acquired at 800 MHz at 37°C ...... 35

2.7 Backbone amide region of '^N-'H HSQC spectrum of the uniformly

15-N-labeled R163K PI-PLC acquired at 800 MHz at 37°C ...... 36

2.8 Backbone amide region of ’^N-'H HSQC spectnun of the ‘^N-glycine

labeled WT PI-PLC acquired at 800 MHz at 37°C ...... 38

XV 2.9 Backbone amide region of *^N-'H HSQC spectrum of the *'N-leucine

labeled WT PI-PLC acquired at 800 MHz at 37°C ...... 39

2.10 ‘^C-'H HSQC spectra o f‘^C-histidine labeled WT PI-PLC (A),

H32A (B), and H82A (C) mutants at 37°C and pH 7.0 ...... 42

2.11 '^C-’H HSQC spectra of ‘^C-labeled histidine residues of WT PI-PLC

at different pHs ...... 43

2.12 NMR titration curves from proton chemical shifrs for histidine residues

of PI-PLC: (A) WT enzyme, (B) D33N mutant, and (C) D274N mutant ...... 44

2.13 Arginine side chain (®NH) region of ‘^N-*H HSQC spectra of uniformly

'^N-labeled PI-PLC: (A) WT; (B) R69D; (C) R69D + 8 mM Sp-DHPsI;

(D) WT + 10 mM 2-MeO-DHPI...... 49

2.14 Backbone amide region of '^N-'H HSQC spectrum of the uniformly

'^N-labeled R69D PI-PLC in the presence of 8 mM Sp-DHsPI acquired

at 800 MHz at 37°C ...... 50

3.1 Activation of R69D PI-PLC by earth-alkaline metals ...... 60

xvi 3.2 Activation of R69D PI-PLC by transition metals ...... 60

3.3 pH-activity profile of R69D PI-PLC without any metal present

(solid line, dark circles) and with 0.8 mM CaCh (dashed line,

open circles) ...... 62

3.4 Arginine side chain (^NH) region of '^N-'H HSQC spectra of uniformly

‘“N-labeled PI-PLC: (A) WT; (B) R69D; (C) R69D + 1 mM CaCh;

(D) WT + 5 mM CaCh...... 64

3.5 Backbone amide region of ‘^N-‘H HSQC spectrum of the uniformly

‘^N-Iabeled R69D PI-PLC in the presence of 5 mM CaCL acquired

at 800 MHz at 37°C ...... 65

3.6 Activation of D33N/R69D PI-PLC by magnesium and calcium ...... 68

4.1 Bacterial PI-PLC and RNase A: a mechanistic analogy ...... 72

4.2 Reactions typically used for the chemical modification of cysteine ...... 75

4.3 Comparison between the side chains of arginine, lysine,

and modified cvsteins ...... 80

X V ll 4.4 (A) Native-PAGE (20%) and (B) lEF (pH 4.0 - 6.5) gels of PI-PLC ...... 82

4.5 Possible arrangements for the Arg69’s bidentate interaction

with the substrate and other components of the active site during catalysis 87

4.6 Activation of R69A and R69G mutants by guanidine chloride ...... 91

5.1 Classification of the transition states in the phopsphoryl transfer

reactions, based on changes in the bond order to phosphorus atom

relative to the ground state ...... 94

5.2 Comparison of imidazole-catalyzed reactions of ribosyl and inositol

phosphodiesters ...... 100

5.3 Structures of aryl inositol phosphates and thiophosphates, used

in the LFER studies...... 102

5.4 ^ ' P-NMR spectra of the representative time courses

of the imidazole-catalyzed transestérification of aryl inositides ...... 104

5.5 Time courses of the imidazole-catalyzed transestérification

reactions of (A) phenol inositol phosphate and (B) 3,5-dichloropheno 1

inositol phosphorothioate ...... 10^

xviii 5.6 Brensted plots of the imidazole-catalyzed transestérification of aryl inositol

phosphates and phosphorothioates ...... 107

5.7 Bronsted plots of the enzymatic transphosphorylation of aryl inositol

phosphates and phosphorothioates catalyzed by WT PI-PLC ...... 107

XIX ABBREVIATIONS

ID - one-dimensional

2D - two-dimensional

AA - acetylamidine

EA- ethylamine

CAA - chloroacetylamidine

Da - Dalton

DHPC - diheptanoyI-5H-giycero-3-phosphocholine

DOsPI - 1,2-dioctanoyI-oxypropane-3-(thiophospho-1 D-wyo-inositol)

DPPI- dipalmi toy Iphosphatidy linosito 1

DPPsI - 1,2-dipalmitoyl-s«-glycero-3-thiophospho-1 -myo-inositol

DTNB- 5,5'-dithiobis(2-nitrobenzoic acid)

EDTA - ethylenediaminetetraacetic acid

GndHCl - guanidine chloride

Gro-PI - jM-glycero-3 -phospho-1 D-wyo-inositol

HEPES - N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid

HSQC - heteronuclear single-quantum coherence

IP - inositol 1-phosphate

XX IcP inositol 1,2-cycIic phosphate

IcPs inositol 1,2-cycIic thiophosphate

IPTG isopropyl-p-D-thiogalactoside

LFER linear free energy relationship

MBS 2-(N-morpholino)ethanesuIfonic acid

MOPS 4-morpholinepropanesuIfonic acid

NMR nuclear magnetic resonance

NOESY nuclear Overhauser enhancement spectroscopy

PA propylamine

PAGE polyacrylamide gel electrophoresis

PI phosphatidylinositol

PIP phosphatidylinositol 4-phosphate

PIP: phosphatidylinositol 4,5-diphosphate

PI-PLC phosphatidylinositol-specific phospholipase C

RNase A bovine ribonuclease A

SDS sodium dodecyl sulfate

TOCSY total correlation spectroscopy

Tris 2-amino-2-hydroxymethyl)-1,3-propanediol

T.S. transition state

WT wild type

XXI CHAPTER I

INTRODUCTION

Life, as we know it, is sustained through complex cascades of chemical reactions

catalyzed by specific enzymes, and any interruption of the catalytic network may be fatal

for the living organism. Mutations impairing catalytic capacity of enzymes were

implicated in a variety of diseases, from various metabolic dysfunctions (Erlandsen et al.,

1997) to cancer (Maehama and Dixon, 1999). Enzymology, as a branch of biochemistry,

has long become much more than academic subject, and now it has broad applications in

medicine, pharmacology, molecular biology, chemical engineering, and food processing, among others. But no matter how far-reaching applications of enzymatic studies have become, “the most fundamental problem of enzymology is the mechanism of action of enzymes, and how to accoimt for the astonishingly high and specific catalytic activity in terms of their chemical structure” (M. Dixon, (Dixon and Webb, 1979))

This work focuses on the catalytic mechanism of phosphatidylinositol-specific phospholipase C (PI-PLC) (EC 1.1.4.10) from insect parasite thuringiensis. In the introduction I will summarize importance of this family of enzymes, our current

knowledge about their structure, regulation, and mechanism, and will rationalize our

choice for the specific subject of the research. Subsequent chapters will be devoted to

specific problems in the mechanism of catalysis and/or to particular methods of

investigation.

1.1 Mammalian PI-PLCs

Eukaryotic PI-PLCs are ubiquitous, large multi-domain proteins with molecular weight ranging from 85 to 150 kDa. There are three families o f mammalian PI-PLC, which differ in the ways they are regulated (Rhee and Bae, 1997; Rhee and Choi, 1992):

P, y, and 5. Protein originally designated PI-PLC a was later shown to be a disulfide isomerase (Katan, 1998). Recently, a completely new type of PI-PLC (originally identified as an effector of Ras) has been described in C. elegans (Shibatohge et al.,

1998), but it is not yet clear how universal this type o f the enzyme is.

1.1.1 Structure

Primary structures of all PI-PLCs known to date have two conserved regions which were originally designated as X and Y domains, respectively (Figure 1.1). As mutagenesis and functional studies have suggested (Simoes et at., 1995; Smith et al., 1994) and crystal structure has confirmed (Essen et al., 1996), X and Y domains form the catalytic core of the enzyme. Various regulatory domains either flank the catalytic center or are inserted PI-PLC-5 \/

PH domain EF-hands Catal^c C2 domain <20 «.a 140 a.a. •260 «.a 1 » «.a.

PI-PLCH5

G-protein binding domain P.Tyr

PI-PLC-y

PI-PLC Bacillus sp. -T

Figure 1.1 : Domain architecture of mammalian PI-PLCs

RCOO RCOO

p=o PI-PLC PI-PLC hat OR slow

HO OR' HO HO OR’ Oiacylglycerol

Figure 1.2: Reactions catalyzed by PI-PLCs:

R', R“ = P0 3 "‘, R^ = H - PfPi, preferred substrate of the mammalian PI-PLC;

R‘ = PO3"', R~, R^ = H - PIP, substrate of the mammalian PI-PLC; R’, R~, R^ = H - PI, substrate of both bacterial and mammalian enzymes; R', R“ = H, R" = glucoseamine - glycosylated PI, substrate of the bacterial PI-PLC. 3 between X and Y domains. Well-defined domain organization of PI-PLC allowed studies some of its regulatory domains long before the structure of the catalytic core became available (Garcia et al., 1995; Pascal et al., 1994; Yoon et al., 1994). Originally published structure of PI-PLC 51 (Essen et al., 1996) represented minimal catalytically active fragment, which included two EF-hands, X, Y, and C2 domains. EF-hands and C2 domain make numerous contacts with each other, thus holding catalytic domain together.

Structure of the catalytic domain is remarkably similar to earlier published structure of

PI-PLC from Bacillus cereus (Figure 1.3) and comprises a slightly distorted a/p barrel, a/p Barrel (also know as TIM-barrel, from the name of the first know member, triosphosphate isomerase) family of proteins includes large number of extremely functionally diverse proteins (for a review, see (Reardon and Farber, 1995)).

Structure of the active site with mvo-inositol 1,4,5-triphosphate (IP3-1,4,5) bound

(Figure 1.4) provided a structural basis for the catalytic roles of several amino acids

(Essen et al., 1997a), enzyme’s preference for the multi-phosphorylated substrate (Rhee et al., 1989), and an essential role of calcium ion for the catalysis (Grobler and Hurley,

1998). Strong interaction of Glu-341 with 3-OH of inositol explained the fact that PI phosphorylated at that position is not a substrate of PI-PLC (Rhee et al., 1991).

1.1.2 Function and regulation

Mammalian PI-PLC converts PI and phosphorylated PI into diacylglycerol

(DAG) and inositol phosphate (Figure 1.2). Both products of the reaction are well- established secondary messengers in numerous signal transduction pathways. DAG transmits its signal through activation of protein kinase C, while inositol phosphates induce release of calcium ions from endoplasmic reticulum (Lee and Rhee, 1995). In

vitro some amount of the reaction intermediate, inositol 1,2-cyclic phosphate (IcP), is

released, but physiological role of IcP, if any, is not clear (Kim et al., 1989).

One of the most amazing features of mammalian PI-PLCs is the number and

variety of signal transduction pathways these enzymes are involved in: from cell

proliferation to apoptosis, from neuronal signaling to muscle contraction, to name just a

few (Berridge, 1987a; Berridge, 1987b; Berridge, 1993; Berridge, 1995; Berridge and

Irvine, 1984). This versatility of PI-PLC enzymes makes them a very attractive target for

the drug development (Bruzik, 1997).

PI-PLC isozymes are activated by trimeric G-proteins (P family) and by tyrosine

kinase-associated receptors (y family). Until very recently it was believed that PI-PLC 5

is only regulated by calcium concentration and serves for non-specific amplification of

the secondary signal (Allen et al., 1997). This hypothesis proved to be wrong when it was

shown that Ô isozyme is activated by a new type of GTP-binding protein named Gh

(Katan, 1998).

While regulation and activation of PI-PLC are well documented and extensively

studied, the way(s) information from a regulatory domain is transferred to the active site

is not known in most cases. Probably the only example is an implication of the flexible

loop between X and Y domains in PI-PLC 51 in the regulation through restricting substrate’s access to the active site (Williams and Katan, 1996). NOTE TO USERS

Page(s) not included in the original manuscript and are unavailable from the author or university. The manuscript was microfilmed as received.

This reproduction is the best copy available.

LJMI B

Figure 1.3: Crystal structures of the bacteria! PI-PLC from Bacillus cereus (A) and truncated (Al-132) mammalian PI-PLC ÔI (B). Structure of the mammalian enzyme includes catalytic domain (bottom; a/(i barrel), C2 domain (top right; p-sandwich), and two EF-hand domains (top left; hclix-loop-hclix). B’ :B'

DAG DAG O «

RO­ OM

Figure 1.4; General acid/base mechanism proposed for PI-PLC: general base activates 2- OH of inositol for the nucieophilic attack; general acid protonates the leaving group, facilitating its departure; positively charged residue (or a metal ion) stabilizes negative charge of the pentacoordinate transition state.

1.1.3 Mechanism

Unlike regulation and roles in signal transduction, mechanism of eukaryotic PI-

PLC enzymes received much less attention. This is largely explained by their relatively high molecular weights and by difficulties to ensure complete activation of the enzymes in vitro. Recently, several published structures of PI-PLC Ô1 (Essen et al., 1996; Essen et al., 1997b), along with some functional (Wu et al., 1997; Zhou et al., 1999) and mutagenesis studies (Ellis et al., 1998) have provided a significant insight into the mechanism of the eukaryotic enzymes. Comparison with better mechanistically studied bacterial enzymes (Hondal et al., 1998) has benefited our understanding of both PI-PLC classes, but also it has brought along a controversy regarding exact roles of specific histidine residues in the catalysis (Heinz et al., 1998). Asp343 GIU390 A ArgM9 T O Asn312 V Lys438R O r~

His356 c r Lys440 7yr551

Figure 1.5; Schematic representation of the active site of mammalian PI-PLC 51 (adopted from (Essen et al., 1996)).

Overall, it appears that all families of the mammalian enzymes employ the same catalytic mechanism, as judged by strict conservation of the key amino acids in the X and

Y domains of all known enzymes (Heinz et al., 1998). Chemically, catalysis by mammalian and bacterial enzymes is absolutely the same (Figure 1.4), the difference comes only from identity of the amino acids participating in catalysis and from the presence of calcium ion in mammalian enzymes. In the first step, 2-OH of inositol, activated by a general base, attacks phosphate to form inositol 1,2-cyclic phosphate. A general acid protonates the leaving group, DAG. In the second step general acid-base pair switches its roles to hydrolyze IcP to inositol 1-phosphate. Negative charge on the phosphate is stabilized by a calcium ion during catalysis. Additionally, calcium ion was

proposed to lower the pKa of inositors 2-OH. Role of the general acid in the first step

was assigned to histidine-356 based on structural and mutagenesis information (Ellis et

al.. 1998; Essen et al., 1996). Identity o f the general base is still unclear, but Glu-341 and

Glu-390 are very strong candidates for that role (Ellis et al., 1998). Position of histidine-

311 (analog of histidine-32 in the bacterial enzyme) precludes it to act as the general base

in the catalysis, instead it appears to stabilize the negative charge and/or to protonate the pentacoordinate transition state (Heinz et al., 1998).

1.2 Bacterial PI-PLC

Bacterial PI-PLCs are relatively small (ca. 30-35 kDa), mono-domain proteins found mostly in pathogens and parasites of higher organisms. Bacterial species with identified PI-PLC enzymes include members of genera Bacillus, Lysteria,

Staphylococcus, and Streptomyces (Bruzik and Tsai, 1994). It was thought that all bacterial enzymes were metal-independent, but one of the two recently described enzymes from Streptomyces antibioticus (Iwasaki et al., 1998) was shown to be calcium- dependent while being in the size range of “regular” bacterial enzymes. This finding has interesting implications about evolution and origin(s) of both pro- and eukaryotic PI-PLC enzymes.

1.2.1 Structure

Structurally, bacterial enzymes are equivalent of the catalytic domain of the mammalian enzyme (Figure 1.3), although sequence similarity is very limited: only about

1 0 30% at the N-terminus, which corresponds to the X domain in the mammalian enzyme

(Bruzik and Tsai, 1994).

The most significant difference between eukaryotic and bacterial PI-PLCs is observed in the active site (Figure 1.6). Bacterial enzyme does not have calcium ion bound in the active site, guanidine group of arginine-69 occupies that position.

.A.dditionally, His32 is located in a perfect position to abstract the proton from 2-OH of inositol. Side chains o f Lysl 15 and Argl63 are in close proximity to 4-OH and 5-OH groups of inositol, while Asp 198 completes the recognition of the inositol headgroup by hydrogen bond to 3-OH.

Arg69 Asp67 O GIU117 O Asp198 N Asp33 Lys115 7 7/ 'j : Asp274 —N \ 29 O

" S J > - H is 3 2 Arg163 HO

HÎS82 < x ^

Tyr200

Figure 1.6: Schematic representation of the active site of bacterial PI-PLC fi-om Bacillus cereus (adopted fi^om (Heinz et al., 1995)).

Il 1.2.2 Function and specificity

Since bacterial membranes do not contain any PI, flmction(s) for the bacterial PI-

PLCs should lie outside the host cell. Indeed, in case of the enzyme from Listeria monocy’togenes, PI-PLC was shown to be indispensable for the virulence of the

microorganism (Camilli et al., 1991). It is reasonable to assume that other bacterial PI-

PLCs are utilized in similar fashion by their producers (Songer, 1997).

Unlike mammalian enzymes, which accept both (poly)phosphorylated and unphosphorylated PI (albeit with different efficiency), bacterial PI-PLCs only cleave the latter (Bruzik and Tsai, 1994). Unique feature of the bacterial enzymes is their ability to release proteins attached to membranes through glycosylated phosphatidylinositol (GPI)

(Stieger and Brodbeck, 1991).

1.2.3 Mechanism

High stability, low molecular weight, availability in high quantities (including from commercial sources), and absence of the regulatory domains made bacterial enzymes the preferred subject of the mechanistic studies early on (Bruzik and Tsai,

1994). Most of the literature on the subject is dealing with the enzymes from Bacillus cereus and B. thuringiensis, which differ only by eight non-conserved amino acids

(Ikezawa et al., 1976; Lechner et al., 1989). Although there are some minor differences in the properties of these two proteins, they are indistinguishable mechanistically.

Therefore, clear distinction between data from the two will be done only when this is absolutely necessary.

12 Early stereochemical studies (Bruzik et al., 1992) and analysis of the products and

intermediates of the enzymatic reaction (Volwerk et al., 1990) had brought forward the

proposal for the general acid/base two step mechanism resembling that of the RNase A*

(Blackburn and Moore, 1982; Richards and Wyckoff, 1971) and consisting of three main

elements (Figure 1.4). First, 2-OH of inositol, in p-position to the phosphate, is activated

for the nucieophilic attack on phosphate by a general base. Second, departure of the

leaving group (DAG) is facilitated by a general acid. Third, a positively charged group stabilizes negative charge on the phosphate. First step produces inositol-1,2- cyclophosphate, an intermediate which is slowly hydrolyzed in the second step. In the second step, a water molecule occupies the place of the DAG in the active site and the roles of the general acid and the general base are reversed.

Further structural (Heinz et al., 1995; Heinz et al., 1996), mutagenesis and functional studies have allowed to identify catalytic residues and to study their functions and interactions. Stereochemical data with phosphorothioate analogs of PI has provided unequivocal evidence of strong interaction of arginine-69 with pro-S oxygen of the phosphate group (Hondal et al., 1997b). Catalytic potency of histidine-82 and aspartate-

33 is rescued by substrates with good leaving groups, thus implicating this diad as the general acid in the catalytic mechanism (Hondal et al., 1997a; Hondal et al., 1998). Most recently, extensive analysis of the thio-effects on the catalysis has identified arginine-69, aspartate-33, and histidine-82 as a unique bifunctional catalytic triad (Kubiak et al.,

1999).

‘ Detailed mechanistic comparison between PI-PLC and RNase A is given below in this Introduction and continued throughout most of the subsequent chapters. 13 Structural arrangement of aspartate-274, histidine-32, and 2-OH of inositol carries

a striking similarity to the catalytic triads of serine proteases (Dodson and Wlodawer,

1998). Although additional evidence for the general base function of the His-32—Asp-

274 diad is very limited at best (Gassier et al., 1997; Hondal et al., 1998), the above mentioned structural similarity and their significant contribution to the catalysis leave little doubt about the pair’s role in the catalytic mechanism.

^ p 3 3 H2N + N- -H'-Q - Q--M À ri|^His82 O His32 R Asp274— )—A \ \ / i X A O" H-N^N- -H-'O T-P V O H \1/ C-2 O C-1

Figure 1.7: Current understanding of the active site residues’ participation in the catalysis by bacterial PI-PLC.

Current status of our imderstanding of the catalytic mechanism by PI-PLC is summarized in Figure 1.7. Whereas most of the available data fit very well into the given scheme, there are quite a few questions remained unanswered about the details of the mechanism. The subsequent chapters of this work address many of those questions, both experimentally and theoretically, giving deeper understanding of the catalysis by PI-PLC and of the phosphoryl transfer reactions (both enzymatic and non-enzymatic) in general.

14 1.2.4 Comparison with RNase A

Ribonuclease A (RNase A; EC 3.1.27.5) is one of the most studied enzymes with almost eight decades of extensive research devoted to various aspects of its structure and function (Blackburn and Moore, 1982; Raines, 1998; Richards and Wyckoff, 1971).

RNase A was and still is the favorite object of benchmark work in the folding, stability, and chemistry of proteins; in enzymology; and in molecular evolution.

A resemblance in the catalytic mechanism for PI-PLC and RNase A was essentially inevitable due to high similarity in both the substrate structures of these two enzymes and the nature of the catalyzed reactions (Bruzik and Tsai, 1994; Hondal et al.,

1997c). A comparison between two enzymes catalyzing similar reactions is, undoubtedly, beneficial for the understanding of both enzymes. In the particular case of RNase A and

PI-PLC, vast amount of knowledge about the former could be effectively used to understand data being acquired for the latter. Conversely, information about PI-PLC’s mechanism might be useful in addressing still unsettled questions (Breslow and

Chapman, 1996; Herschlag, 1994) and unproved theories (Gerlt and Gassman, 1993) in the mechanism of RNase A.

15 CHAPTER 2

NMR STUDIES OF BACTERIAL PI-PLC

2.1 INTRODUCTION

2.1.1 NMR o f large proteins

Nuclear magnetic resonance (NMR) studies of biological macromolecules had gone a long way from first published 'H NMR spectra of proteins and nucleic acids

(Jacobson ei al., 1954; Jardetzky and Jardetzky, 1957; Saimders et al., 1957). Last three decades have seen a tremendous progress in both hardware and methodology of protein

NMR (reviewed in (Bax, 1989; Bax, 1994; Bax and Grzesiek, 1993; Clore and

Gronenbom, 1998)): high-field (up to 750 and 800 MHz) magnets are becoming widely available, stable isotope (‘^ , *^C, and ^H) labeling techniques are developed almost to perfection, new multi-dimensional methods are being constantly developed and improved. One of the latest developments in the field is application of liquid crystal media to measure heteronuclear dipolar coupling (Tjandra and Bax, 1997; Tjandra et al.,

1997), which allows for both improvement in structure quality and increase in proteins’ size available for structure determination in solution. All of the developments mentioned

16 above has simplified structure determination and has significantly increased the upper

size limit of proteins and protein complexes amenable to NMR.

PI-PLC from Bacillus thuringiensis has a molecular weight of 34.5 kDa (298

amino acids). Proteins of this size are still quite challenging for determination of the

solution structure, but availability of high field (600 and 800 MHz) NMR spectrometers

in combination with stable isotope labeling should be sufficient to solve the structure by

NMR, provided there are no solubility and/or aggregation problems.

At the time these studies had begim, there were no structures of any PI-PLC

available. Therefore, one of my goals was attempt to solve the structure of the bacterial

PI-PLC by NMR. Unfortunately, soon after conditions for the NMR studies of this enzyme were worked out, crystal structure of B. cereus PI-PLC, which differs from B. thuringiensis PI-PLC only by eight non-conserved amino acids (Volwerk et al., 1989b), was published (Heinz et al., 1995). This development made the pursuit of the solution structure of mostly academic interest. On the other hand, our experience with the NMR characterization of the enzyme provided us with relatively simple method to monitor structural integrity of the mutant proteins in our functional studies (Hondal et al., 1997b;

Hondal et al., 1998). Furthermore, during an investigation of the structure-function relationships of PI-PLC we have come upon several issues (as described below) which are best addressed with NMR methods.

2.1.2 pH-activity profiles of WTand mutant PI-PLC.

WT PI-PLC has imusually broad pH profile with apparent pKaS of approximately

3.5 and 8.5 (Hondal, 1997; Zhao, 1996). This fact does not agree very well with the

17 proposal of two histidine residues acting as general acid/base pair in the mechanism of

the enzyme action since pKa of a free histidine in solution is about 7.0 (Markley, 1975). It

is not unusual for the amino acid side chains in the enzyme active sites to change their

intrinsic pKa’s significantly, especially upon binding of the respective substrates (or substrate analogs) (Perez-Canadi 1 las et al., 1998; Yu and Fesik, 1994). In case of bacterial PI-PLC, a dramatic lowering of the one of the histidine’s pKa seems very unlikely, since both of them are closely associated structurally and functionally with aspartic residues, forming catalytic diads His-32—Asp-274 and His-82—Asp-33 (Heinz el al., 1995; Hondal et al., 1997a). Alternatively, broad pH profile could be determined by more complex phenomena than pFQs of the general acid/base in the catalysis. One possibility is that the general base diad remains functional as long as its carboxylate component stays unprotonated.

Even more intriguing are the changes to the pH profile coursed by mutations of

Asp-33 and Asp-274 to asparagine (Hondal, 1997; Zhao, 1996): while D33N mutant displays pH-activity profile essentially identical to the WT’s, D274N mutant has quite narrow pH profile with its basic limb shifted by more than 2 pH units to the lower pH.

Such pattern would be expected if the pKa of a general acid were affected, but Asp-274 is a part of the general base diad. Once again, this paradox could be explained by a complex nature of the pH profile and/or by the fact that D274N mutation affects pKa of its diad partner to a lesser extent than another component(s) of the active site.

In attempt to correlate pKaS of the active site histidines with the changes in the pH-activity profiles, determination of the histidine pKaS in the WT enzyme along with

D33N and D274N mutants has been undertaken.

18 2.1.3 “Hydrophobic ” activation o f PI-PLC.

Like many lipases and phospholipases, bacterial PI-PLC has a preference for its substrate in the form of micelles or vesicles (Volwerk et al., 1994), but unlike, for example, phospholipase A: (Jain and Berg, 1989), PI-PLC is only modestly activated by the aggregated substrate. In contrast, Gro-PI (PI without hydrophobic acyl chains) is much worse substrate than PI: is lower ca. 2000 fold and Km is higher ca. 900 fold with this substrate (Kubiak, 1999; Kubiak et al., 1999). It is not unusual for an enzyme’s activity, particularly for one working on a polymeric substrate, to be affected by a part of the substrate distal to the catalytic site. Such phenomena were demonstrated, among other cases, for RNase A (Raines, 1998) and, probably in the purest form, for type I ribozyme

(Narlikar and Herschlag, 1998) catalysis. The unusual feature of the bacterial Pi-PLC in that respect is the fact that it does not have well-defined binding site for the hydrophobic part of its substrate. Instead, it has a short hydrophobic helix (residues 42-48) and a hydrophobic loop (residues 237-243), forming a “hydrophobic ridge” around the active site, which was suggested to participate in interaction with the membrane and/or with the acyl chains of the substrate (Heinz et al., 1998; Heinz et al., 1995).

Combination of structural and functional information available to the date allows us to suggest at least two possible models of the enzyme’s activation by the hydrophobic acyl chains of the PI (Figure 2.1). In each model interaction of “hydrophobic” (with acyl chains present) substrate with the enzyme is compared to that of “hydrophilic” (without acyl chains) substrate. In the first model (Figure 2.1, A), hydrophobic part of PI contributes to the catalysis through introduction of strain in the scissile bond. Such

19 A B

Figure 2.1: Two potential models for the activation of the bacterial PI-PLC by hydrophobic part of its substrate: (A) acyl chain(s) of PI “clash” with the hydrophobic ridge, producing strain in the scissile bond; (B) interaction with the acyl chains produces conformational changes, which are translated to the catalytic residues (represented by red triangles) in the active site. Complete analysis o f both models is given in the main text.

arrangement necessitates very strong binding of the inositol headgroup, with part of the binding energy being transferred to the catalytic power of the enzyme. In the second model (Figure 2.1, B), interactions bef veen hydrophobic components of the enzyme and substrate cause rearrangements in the active site (including, but not necessarily limited to, catalytic residues) resulting in the better binding and improved catalysis.

A conformational change is the most significant difference between these two models: the second model requires it, while first one is based on the assumption of quite rigid protein structure. Furthermore, second model complies much better with requirements for an effective catalysis: upon release of diacylglycerol, affinity to the inositol part of the substrate dramatically decreases, allowing for quick and easy release of the second product, IcP.

2 0 Existing experimental evidence in the relation to the models under consideration

could be described as contradictory. On the one hand, crystal structures of the free

enzyme and enzyme with /w>o-inositol or glucosamine-inositol bound do not have any

significant differences (Heinz et al., 1995; Heinz et al., 1996), which favors model I. On

the other hand, PI-PLC has very low affinity to hydrophilic versions of its substrate, Gro-

PI and IcP (Kubiak, 1999; Zhou et al., 1997b). Activation o f PI-PLC toward water-

soluble substrates by lipid-water interface (Zhou et al., 1997a; Zhou et al., 1997b) and by

organic solvents (Zhou and Roberts, 1998) also speaks in the favor of a conformational

change (model II).

In order to distinguish between two proposed models, interaction of several

substrate analogs with PI-PLC was studied by heteronuclear NMR spectroscopy. All

analogs in this study have hexanoyl acyl chains. Very low affinity of hydrophilic

substrates (IP, IcP, and Gro-PI) makes their use for NMR binding studies virtually

impossible.

2.2 MATERIALS AND METHODS

Trypton and yeast extract were from Difco Laboratories; DiO, TMSP-d», ’^C- and

‘^N-labeled amino acids were from Cambridge Isotope Laboratories; all other chemicals were of the highest quality available commercially.

21 E. coli competent cells were prepared according to procedure of Chung et al.

(Chung et al., 1989) and were stored at -70°C. No significant decrease in cells’ competency was observed for at least 6 month.

All DNA-modifying enzymes were fi’om New England Biolabs. Taq DyeDeoxy terminator thermocycle sequencing kit was fi-om Applied Biosystems. Standard DNA manipulations were done according to published procedures (Maniatis et al., 1982) and/or according to supplier’s manuals.

Sp-l,2-dihexanoyl-sn-glycero-3-thiophospho-l-/wyo-inosotol (Sp-DHPsI), 1,2- dihexanoyl-s«-glycero-3-phospho-2-deoxy-/nvo-inositol (2-deoxy-DHPl), and 1,2- dihexanoyl-s/2-glycero-3-phospho-2-0-methyl-/Nyo-inositol (2-methoxy-DHPI) were provided by Dr. Karol S. Bruzik’s lab (University of Illinois at Chicago).

Construction and functional characterization of all PI-PLC mutants used in this chapter are given in the subsequent chapters, in the previously published papers (Hondal et al., 1997b; Hondal et al., 1998), and/or in the dissertations of others from this lab

(Hondal, 1997; Riddle, 1997; Zhao, 1996).

2.2.1 Protein expression

During the course of these studies several systems were used for overexpression of PI-PLC in E. coli:

pHN1403 vector (originally a gift from Dr. F. Dahlquist, University of

Oregon) in E. coli strain MM294. This system was modified and described in

detail by S. Riddle (Riddle, 1997);

2 2 pET2 lb vector (Novagen) carrying plcbt gene preceded by STII signal

sequence in E. coli strain BL21(DE3). This vector was constructed by R. J.

Hondal (Hondal, 1997);

Protein expression from the above two systems was described before (Hondal

et at., 1997b).

pET21b vector (Novagen) carrying plcbt gene without signal sequence in E.

coli strain BL21(DE3).

Subcloning PI-PLC gene without signal sequence. Both empty pET21b plasmid and the one containing plcbt gene with the signal sequence were cut with Nhel and Sail restriction enzymes. Reaction with the empty plasmid was additionally treated with alkaline phosphatase. Both plasmid and gene were gel-purified with Geneclean kit (Bio

101, Inc.). Ligation reaction was done at 12°C with T4 DNA ligase for 12 hours. One- half of the ligation reaction was transformed into E. coli XL 1 Blue (Novagene) competent cells. Successful insertion of the gene was confirmed with restriction digest and DNA sequencing.

Protein overexpression. E. coli BL21(DE3) carrying pET21 b-p/c6r plasmid was grown in LB media (typically 4 liters) with 100 mg/L of ampicillin at 37°C until ODeoo ~

1.0, then IPTG was added to 1 mM total. Expression continued for additional 6-7 hrs at room temperature. Cells were collected by centrifugation and stored at -20°C until further use.

23 2.2.2 Protein purification

Frozen cells were resuspended in ice-cold 20 mM Tris buffer (pH 8.5) with I mM

EDTA and were broken by sonication. Cell debris were removed by centrifugation and

supernatant was loaded on pre-equilibrated DEAE-sephacryl (Pharmacia) column (-150

ml bed volume). This and all subsequent purification steps were done in a cold room.

Column was washed extensively with the loading buffer followed with 3-4 bed

volumes of 50 mM NaCl in 20 mM Tris (pH 8.5). Protein was eluted with gradient of 50-

200 mM NaCl in 15 ml fractions. Fractions were analyzed by UV and SDS-PAGE. As a

rule, PI-PLC was the major peak in the second half of the gradient.

Fractions containing PI-PLC were pooled and protein precipitated by (NH 4)2S0 4

(-80-85% of saturation). Protein precipitate was redissolved in 6-8 ml of 20 mM Tris and

loaded on Sephadex G-lOO or G-75 (Pharmacia) gel-filtration column (bed volume -600 ml). 5 ml fractions were collected and analyzed by UV and SDS-PAGE. PI-PLC’s peak

(as a rule, second peak in the elution profile) was pure in its later two thirds.

If purity of the protein was not satisfactory after the gel-filtration column, more pure fractions were pooled, NaCl was added to 1.5 M and loaded on a pre-equilibrated

Phenyl-Sepharose column (ca. 10 ml bed volume; Pharmacia). Column was washed with

5-10 bed volumes of the loading buffer and protein was eluted with 20 mM Tris, pH 8.5.

Fractions containing pure protein were pooled and extensively dialyzed against 1 mM

HEPES buffer. After final dialysis protein was lyophilized and stored at -20°C, unless any special preparation(s) was needed. Typical yields were in the range 25 - 50 mg of pure protein from 1 L of cell culture.

24 2.2.3 Uniform and specific labeling of PI-PLC with and

Uniform labeling with was achieved by protein expression in standard M9 media (Maniatis et al., 1982), in which ammonium chloride was replaced by ammonium chloride (>99% isotope enrichment. Isotech or Cambridge Isotope

Laboratories). After induction with IPTG, protein expression continued at room temperature for 9-10 hrs. Otherwise expression was no different from the one in LB media.

Specific labeling with amino acid(s) carrying either ‘^C or isotopes was accomplished by expressing PI-PLC in the synthetic rich (SR) media without resolving to auxotrophic strains of E. coli. Labeled amino acids were added to the media at the same time as IPTG. Otherwise protein expression was no different from the one in the LB media.

SR media was prepared as follows (with the amino acid of interest excluded)

(Muchmore et al., 1989):

The following was dissolved in 950 ml of water: alanine 0.50 g, arginine 0.40 g. aspartate

0.40 g, cysteine 0.05 g, glutamine 0.40 g, glutamate 0.65 g, glycine 0.55 g, histidine 0.10 g, isoleucine 0.23 g, leucine 0.23 g, lysine HCl 0.42 g, methionine 0.25 g, phenylalanine

0.13 g, proline 0.10 g, serine 2.10 g, threonine 0.23 g, tyrosine 0.17 g, valine 0.23 g; adenine 0.50 g, guanosine 0.65 g, thymine 0.20 g, uracile 0.50 g, cytosine 0.20 g, sodium acetate 1.50 g, succinic acid 1.50 g, NH4CI 0.50 g, NaOH 0.85 g, K2HPO4 10.50 g.

After autoclaving the following was added aseptically: 50 ml of 40% glucose, 4 ml of 1

M MgS 0 4 , 1.0 ml of .01 M FeCb, 10 ml of filter-sterilized solution containing: 2 mg

25 CaCl] HzO, 2 mg ZnS 0 4 YHiO, 2 mg MnSO^ H?0, 50 mg tryptophan, 50 mg thiamine,

50 mg niacin, 1 mg biotin, and 100 mg ampicillin.

2.2.4 Sample preparation for NMR experiments

For NMR experiments which did not include any type of titration, residual

concentration of HEPES buffer was used; after final dialysis against 1 mM HEPES

samples were concentrated by Centricon-10 (Amicon) to ensure final buffer concentration no more than 0.1 mM. For the experiments involving any kind of titration, either 50 mM phosphate (for *H and '^C-'H experiments) or 50 mM HEPES (for '^-*H experiments) buffers were used. pH was monitored by the combined microelectrode

(Wilmad) with digital pH/ion meter (Orion Research). pH was adjusted with NaOH

(NaOD) or HCl (DCl) as appropriate.

2.2.5 ID and 2D NMR experiments

Both one-dimensional NMR and two-dimensional NOESY NMR spectra were taken of wild-type and mutant enzymes to determine the structural integrity of the mutants with respect to WT. NMR spectra were recorded on a Bruker DMX-600 NMR spectrometer at 37°C. PI-PLC was redissolved in 99.97% DiO to a concentration of 0.2-

0,4 mM and adjusted to pH 6.8 (direct pH meter reading). Proton and carbon chemical shifts were referenced to intemal TMSP-i/4 standard or to external 2,2-dimethyl - silapentane-5-sulfonate (DSS) set to 0 ppm (Wishart et al., 1995). Two-dimensional

NOESY spectra were acquired with a mixing time of 100 ms. The residual water signal was suppressed by a 3-9-19 pulse sequence with gradients (Piotto et al., 1992; Sklenar et

26 al., 1993). A two-dimensional HSQC (Bodenhausen & Ruben, 1980; Bax et al.,

1990) experiments were conducted using the sensitivity-enhanced method (Kay et al.,

1992) on uniformly and specifically ‘^-labeled samples in 90% H2O/10% D 2O on either

DMX 600 or DMX 800 NMR spectrometers. Prior to Fourier transformations, ID spectra

and F2 dimension of 2D spectra were processed by Gaussian function (GB = 0.1, LB = -3

Hz); FI dimension of 2D spectra were processed by a shifted sine-bell function (SSBl =

8). All spectra were acquired at 37°C.

2.2.6 Determinations of the pKaS o f histidines in WT enzyme and active site

mutants.

Two-dimensional ‘"’C-'H HSQC spectra of proteins labeled with L-histidine (ring-

2-'"C, 99%) at pH values ranging fi'om 4 to 10 were recorded on a Bruker DMX 600

NMR spectrometer at 37 °C. Each sample contained 0.3-0.4 mM enzyme and 50 mM phosphate buffer in 90% H2O/10% D 2O. The D274N mutant was found to be more stable in D2O and thus was kept in 100% D 2O. Assignments of signals from His32 and His82 were made by comparing the spectra of WT PI-PLC with those of the corresponding alanine mutants. Assignments of four other histidines were based on those by Liu et al.

(Liu et al., 1997). The pH dependence of CeH chemical shifts of each titratable histidine residue was fitted to modified Henderson-Hesselbach equation by the least-squares method:

ôobs = Ô ah+(Ô a-Ô ah)(10P”''’‘^)(1 + 1 0 PH-P*^)-', where 5ah and 5a are the chemical shifts of the fully protonated and deprotonated states, respectively.

27 2.2.7 Proteiti-ligand interactions monitored by NMR

In order to ensure an adequate sensitivity and to reduce the experimental time, ail

ligand titration experiments were done on DMX 800 NMR spectrometer (Bruker). Stock

solutions of the substrate analogs were made of the highest concentration possible

(typically 100 mM) so volume of an NMR sample does not change more than 20% during an experiment.

2.3 RESULTS AND DISCUSSION

2.3.1 ID proton, 2D NOESY and HSQC NMR as monitors of global protein

structure

Probably the most important challenge in the analysis of site-directed mutagenesis data is a requirement to distinguish effects from a global structure perturbation and from perturbation of one (or several) amino acid’s frmction(s) (Gerlt, 1987; Knowles, 1987;

Wagner and Benkovic, 1990). One of the approaches to do such a distinction is to solve crystal structures of all or most of the analyzed mutants (Ellis et al., 1998; Gassier et al.,

1997). This approach is quite time and resource consuming and requires a crystal structure of WT protein to be available in the first place. Alternatively, high-field NMR spectroscopy can be used for a qualitative monitoring of mutants’ global structure even if the solution structure or even the total NMR assignment of the protein in question is not available (Hondal et al., 1998; Tevelev et al., 1996; Tsai and Yan, 1991; Vanet al.,

1990). This method generally faster and less expensive compared to crystallography, 28 while it has significant limitations in terms o f protein’s size, solubility, and aggregation

state.

As mentioned before, bacterial PI-PLC has relatively large size for NMR studies.

Besides, it tends to aggregate at the concentrations regularly used for NMR spectroscopy

(i.e. > 1 mM), producing very broad spectra (data not shown). Combination of low protein concentration and high field NMR allowed to obtain NMR spectra of reasonably good quality for the protein of this size. Figure 2.2 shows ID proton spectra of WT and several representative active site mutants. All spectra are very similar in both aliphatic and aromatic regions, as well as their line widths. This clearly indicates that there is very little, if any, structural perturbations introduced by the point mutations. The only notable difference is observed in the amide region (8.5-9.5 ppm) in the spectrum of D274E mutant (Figure 2.2, D). Most signals in that region have disappeared for D274E, while other mutants and WT enzyme have numerous signals there. This can be rationalized in terms of higher flexibility of D274E mutant, which will allow for normally slow exchanging amide protons to be more easily replaced by deuterons. Higher flexibility of this mutant is confirmed by its very low stability toward dénaturation by guanidine chloride compared to WT and most other mutants (Hondal et aL, 1998; Zhao, 1996).

2D NOESY experiments on the same set of proteins (Figure 2.3) further confirm high structural tolerance of PI-PLC to point mutations. Both aromatic-aromatic (lower portion of each spectrum) and aromatic-aliphatic (upper portion) cross-peaks show very similar pattern for all examined proteins, which is an excellent indication that both secondary and tertiary structures are intact. Slight differences in signal intensity between

29 — I— —1— — r — 1 - -* ■ I —I— 8.0 7.0 8.0 ppm 2.0 1.0 0.0

Figure 2.2: One-dimensional proton NMR spectra of WT, H32A, D274A, D274E, H82A, and D33A in DiO at 600 MHz: (A) WT, (B) H32A, (C) D274A, (D) D274E, (E) H82A, and (F) D33A. Sharp signal at 0.0 ppm belongs to TMSP-

30 ••

• • • • ", ^ •• • ■ V ‘ ••

• «m^ • •• m*W • ' # ' # * * • • #*# • • •

^ # « e * • ••

p@#

B #■ - -# • *

- ;Ù:,= t:- .' < *.*• ■ € . * .1 ■ : # & m* « • • • ✓* ■« *

0

ppm ppm

Figure 2.3; Two-dimensional phase-sensitive NOESY NMR spectra of WT, H32A,

D274A, D274E, H82A, and D33A in D 2O at 600 MHz: (A) WT, (B) H32A, (C) D274A, (D) D274E, (E) H82A, and (F) D33A. Mixing time was 100 ms. Detailed conditions are given in the Materials and Methods section.

31 spectra are most likely due to variations in individual protein’s dynamic and/or aggregation properties.

Due to development of highly effective bacterial expression systems (such as pET by Novagen) and availability of relatively cheap '^-enriched salts, uniform labeling of recombinant proteins with has become a very common practice and has been even suggested as a standard procedure for the characterization of mutant proteins by heteronuclear NMR (Venters et al., 1995).

PI-PLC was uniformly labeled with by expressing the protein on the minimal media with ammonia salts as the only nitrogen source. Backbone amide region of

'■'N-'H HSQC spectrum acquired at 600 MHz of the labeled WT enzyme is shown on

Figure 2.4. At least 70% of possible 284 signals' could be identified on the spectrum, which is very high number for the protein of this size without deuteration (Clore and

Gronenbom, 1998). Both signal-to-noise ratio and number of observable peaks significantly increases for the spectrum acquired at 800 MHz (Figure 2.5), while time to obtain the spectrum decreases almost three-fold.

'^N-’H HSQC spectra of both R69D (Figure 2.6) and R163K (Figure 2.7) mutants show high similarity with that of WT enzyme, indicating that protein’s structure can pretty well tolerate dramatic (arginine to aspartate) as well as conservative (arginine to lysine) point mutations.

■ PI-PLC has 298 amino acids, but 13 proline residues do not have the amide proton. Additionally, N- terminal NH^-group is not normally visible due to exchange with solvent. Therefore, maximum number of the amide signals is 284.

32 -105

110 «9

-115

120

w -125

• • • -130

-135

ppm ppm 11

Figure 2.4: Backbone amide region of HSQC spectrum of the unifonnly '^N-labeled WT

PI-PLC acquired at 600 MHz at 37°C. Sample is in 90% H2O/10% D]0, pH 7.5. o e 105

* 110

• CO|Po» 115

120

^»N •.%Oao* «pO w 125 O O A ^ o

% 130

135

ppm 1 I I I 1I I t I I r'I I r~r | r i i i i i i i i r i i i~i i i ' t i i~ ti" i i i i i i i i i r i i , n r t i i i i i ppm 11 10 H

Figure 2.5: Backbone amide region of HSQC spectrum of the uniformly '^N-iabeled

WT PI-PLC acquired at 800 MHz at 37°C. Sample is in 90% H10/10% D 2O, pH 7.5. w -105

<6 -110

-115 % 120 15,N w 125 ^ o * • c . ® 130 % .

135

ppm 1 - 1 - 1 —I » î —I I I I "!■ r I—I—I- i - r - ? ' I—I—I—n ~ i —r—i—i—r~i—i—r—i—i—|—r—i—«—-i—i—i—i—i— —i—i—r- r - r —t—i~ r-^ —i—i—i—r—r ppm 11 10 9 . 8 7 6 'H

Figure 2.6: Backbone amide region of ' HSQC spectrum of the unifonnly -labeled R69D

PI-PLC acquired at 800 MHz at 37°C. Sample is in 90% H20/10% D.O, pH 7.5. 6 105

110 ® / V f®

115

120 15|N

0\u> 125

-130

135

ppm ~T I 1 ■ r I 1 » I I I I - —]—I— I— I— r —1—r—1— I— r - ^ — i— i— i— t - f —i— i— i— i— ;— r —i— i— I’- r - f — i i i | ■ i " i— r ppm 11 10 9 8 7 6 'H

Figure 2.7: Backbone amide region of ‘ HSQC spectrum of the uniformly ' ^N-labeled

R163K PI-PLC acquired at 800 MHz at 37°C. Sample is in 90% H2O/10% D^O, pH 7.5. Overall, ID and 2D NMR spectroscopy has demonstrated that B. thiiringiensis PI-

PLC is able to maintain its overall structure upon point mutation of numerous amino acids in and around the active site. This conclusion is corroborated by data from guanidine chloride-induced dénaturation of WT and mutants (Hondal, 1997; Riddle,

1997; Zhao et al., 1996) as well as by several published crystal structures of the same or similar mutants of B. cereus enzyme (Gassier et al., 1997), which is almost identical to our protein. It appears that if mutants of the bacterial PI-PLC are stable enough to be expressed in soluble form and to be purified, they are going to retain the native structure.

The structural information about mutants of PI-PLC was extensively used in this and other work (Hondal, 1997; Hondal et al., 1997b; Hondal et al., 1998; Riddle, 1997;

Zhao, 1996) as a guide for the interpretation of the kinetic, stereochemical, and other functional data of PI-PLC mutants.

2.3.2 Towards the total NMR assignment of PI-PLC

In the process of total NMR assignment by heteronuclear methods, spectra of the specifically labeled proteins can provide a starting point in the NMR assignment as well as serve as reference points during the sequential assignment (Ikura et al., 1990). Besides, specifically labeled proteins produce very little, if any, signal overlap, allowing for a better assessment of spectra’s quality.

PI-PLC was specifically labeled, in separate experiments, with '^N-glycine and

‘^N-leucine. ‘^N-‘H HSQC spectrum of '^-glycine labeled protein shown on Figure 2.8.

WT enzyme has total sixteen glycines in its sequence, but only 13 signals are observed in the spectrum. This is not very surprising taking into account that glycines tend to be

37 e> o 105

O 110 O

115

•120 15iN

W 125 00

-130

-135

ppm ■ I I I I I - r » I I—I—r - i - T I—p —I—I - 1—r—1—I—i-T —1—I—r - i —r—t—i—i—i—t—i—f“ i' '» i—i—i—i—r—t—i [—t m i r- ppm 11 10

Figure 2.8: Backbone amide region of HSQC spectrum of the '^N-glycine labeled

WT PI-PLC acquired at 800 MHz at 37°C. Sample is in 90% HiO/10% DiO, pH 7.5. 105

110

115

120 15^

125 VOw «9 9 130

136

ppm l-'l-l I I I I T - |“ T-f ■ r T p T- l-'l-T'I- I -T I 1 \ «—T'-1-r I-1-1-1 -T-| T'W T- t I I I l-T-p » I f IT -I' |-T-T-p T-t~r-! ppm 11 10 9 8 'H

Figure 2.9; Backbone amide region of '"'N-'H HSQC spectrum of the '^N-leucine labeled

WT PI-PLC acquired at 800 MHz at 37°C. Sample is in 90% H20/IO% DiO, pH 7.5. located in the flexible structural elements of the protein, NMR signals from which

could broaden up below detection limits. In fact, twelve of the PI-PLC’s glycines are

found in turns and loops (Heinz et aL, 1995). Since glycine signals are the most likely

ones to be absent in the NMR spectra, the above results put lower limits on the total

number of observed backbone amide signals in the uniformly labeled protein at 80%.

This assertion is supported by the spectrum of ‘^-leucine labeled PI-PLC (Figure 2.9).

Signals from all twenty leucine residues are easily identifiable in the spectrum. Since no

leucine-deficient E. coli strain was used for the expression of the labeled protein, some

scrambling of the label was observed (data not shown). Judging from those low intensity

signals in the spectrum, the scrambling was amino acid-specific. One o f the small signals

appears in Figure 2.9 at 129.4 ppm and 8.3 ppm 'H. Comparison with the spectrum of

uniformly labeled protein (Figure 2.5) identifies that signal as the one with the highest

intensity in the whole spectrum. This explains why its height is comparable to the signals

of leucines, even though its '^-enrichm ent must be significantly lower.

Both spectra o f glycines and leucines have one signal each substantially

downfield from its peers (Figures 2.4 and 2.6). Since signals of side chains of active site

histidines are also shifted downfield from the rest of the histidines (see below), there is a

high probability that those unusual glycine and leucine signals belong to the amino acids

in or very near the active site. Two glycine residues, Gly-70 and Gly-83, are very strong

candidates for the signal at 124 ppm and 11.0 ppm 'H. Leu-80 is most likely

responsible for the signal 131 4ppm and 10.5 ppm 'H. Both those signals either move or disappear in the spectrum of R69D mutant (Figure 2.14), which supports their assignment to the vicinity of the active site.

40 Results with the specifically labeled PI-PLC further demonstrate feasibility of the total NMR assignment of this protein and will provide excellent reference points for the

latter. Furthermore, analysis of chemical shifts in the spectra of the specifically labeled proteins has allowed several tentative assignments in and around the active site of the enzyme.

2.3.3 pKaS of the histidine residues in WT PI-PLC and selected active site mutants

As mentioned above, Asp-33 and Asp-274 were suggested to assist in the function of the general acid and general base, respectively. In order to further test their roles in the catalysis and their influence on acid-base properties of the active site histidines, pKaS of histidines in WT enzyme and in two active site mutants, D33N and D274N were determined by 2D heteronuclear NMR spectrosopy.

WT PI-PLC has six histidines in its sequence (Lechner et al., 1989) and '"’C-'H

HSQC of the specifically labeled enzyme contains six well-resolved signals (Figure 2.10,

A). Only one of the signals disappears upon either H32A (Figure 2.10, B) or H82A

(Figure 2.10, C) mutations, allowing for the unambiguous assignment of signals from the active site histidines. Assignments of four other signals were taken from the work on B. cereus enzyme (Liu et al., 1997).

Figure 2.11 shows the representative HSQC spectra of WT PI-PLC as the enzyme is being titrated from pH 4.2 to pH 7.9. The titration curves derived from such HSQC experiments are shown in Figure 2.12 and the pKa values determined from these curves are summarized in Table 2.1. Chemical shifts of His-61 and His-81 were not changing

41 -136

-138

-140 ppm

-136

B -138 " C

-140 ppm

-136

His61 His227 H is8 1 H is 3 2 -138

H is 9 2

H is 8 2 -140 ppm

Figure 2.10: '^C-'H HSQC spectra o f'^C-histidine labeled WT PI-PLC (A), H32A (B), and H82A (C) mutants at 37°C and pH 7.

42 1 1 t :3c 0 i 4 r 138 i j Q ® pH = 7.9 :.Ti

135 (3 ♦ -158 0 ® pH = 7.2 pcm

-136 9 4 -136 13/ 9 ^ pH = 6.9 PPT.

©

■138

pH = 6.4

pH = 4.2

Figure 2.1 1 : HSQC spectra of '^C-labeled histidine residues of WT PI-PLC at different pHs. The arrows show the position of the signal belonging to His-227 as it is being titrated from pH 4.2 to 7.9. Sample is in 50 mM phosphate buffer. Spectra were acquired on Bruker DRX-600 spectrometer at 37°C. 43 90

His82 His227 I

His92 His6l

HisSI

70 4 5 6 7 8 9 10 pH

90

His32

B I His92 His82 His61

HisSI His227 i

4 5 6 8 9 10 pH

90

His82 85

80 His92

His6I 75

HisSl

70 4 5 6 7 8 9 10 pH

Figure 2.12: NMR titration curves from proton chemical shifts for histidine residues of PI-PLC: (A) WT enzyme, (B) D33N mutant, and (C) D274N mutant. 44 pKa Enzyme His32 His82 His92 His227

WT 7.4 7.0 5.6 6 .8

D274N NO* 7.2 <6.5** 7.0

D33N 6.8 6 .2 5.5 6 .8

*H32 was not assigned in D274N mutant

** Due to the mutant’s instability at low pH, only an estimate can be made for this amino acid

Table 2.1. pKa's of titratable histidines in WT PI-PLC and D—»N active site mutants

much in the pH range studied for all three proteins. This is consistent with the crystal structure of the bacterial PI-PLC, in which side chains of these two histidines are buried inside the protein and are not exposed to solvent (Heinz et al., 1995).

The results for the WT enzyme are essentially the same as those recently published for B. cereus PI-PLC (Liu et al., 1997). The greater instability of the D274N mutant prevented the unambiguous assignment of a resonance for its His-32. As a result, only the pKa values of His-82 and His-227 were determined for this mutant, and both pKa values in the mutant were close to those in the WT enzyme. A substantial shift in the pKa's for His-32 and His-82 was observed in the 033N mutant (0.6 and 0.8 pKa units.

45 respectively), while the pKa's o f His-92 and His-227 did not differ from those of WT PI-

PLC.

Only data for His-32 and His-82 are of interest for the discussion, but the fact that

four other histidines do not change their properties upon mutations in the active site

confirms that all observed perturbations are localized to the active site of the enzyme.

Ribonuclease A also utilizes two histidines for the intramolecular cleavage of a

phosphodiester bond with the formation of cyclic phosphate intermediate (Blackburn and

Moore, 1982; Richards and Wyckoff, 1971; Thompson et al., 1995). Its catalytic

histidines. His-12 and His-119, exhibit pKa values of 5.8 and 6.2 (Markley, 1975), which

is consistent with their roles as general base and acid, respectively. In contrast, pKa of the

proposed general acid in the PI-PLC reaction, His-82, is lower than pKa of the proposed

general base, His-32. Those pKaS are more consistent with the proposed roles of the

histidines in the second reaction of PI-PLC, hydrolysis of inositol cyclic phosphate. The role of His-82 as the general acid in the first step is well documented (Gassier et al.,

1997; Hondal et al., 1997a; Hondal et al., 1997c), while evidence for the activation of 2-

OH by His-32 is mostly structural (Heinz et al., 1998; Heinz et al., 1995). The situation is reminiscent of the discussion about the identity of the general base in the mechanism of ribonuclease T, (Steyaert, 1997). RNase T, also has two histidines, His-40 and His-92, in the active site with relatively high pKg values, 7.9 and 7.8, respectively (Inagaki et al.,

1981). His-92 is the general acid in the catalysis (Heinemann and Saenger, 1982), but a glutamic acid, Glu-58, not a histidine, is the general base (Steyaert et al., 1990).

Ambiguity in case of PI-PLC is aggravated by the crystal structure of the mammalian

46 enzyme (Essen et al., 1996), which shows no histidines in the vicinity of inositol’s 2-OH with both active site histidines hydrogen-bonded to the 1-phosphate (Figure 1.5).

pKaS of both His32 and His82 are both lowered by D33N mutation (Table 2.1), with the effect on His82 being slightly bigger. D274N mutation does not affect the pKa of

His82, but it brings dramatic dynamic changes to the side chain of His32 since signal from the latter cannot be found in the spectra. It appears that roles of the aspartates in

His32—Asp274 and His82-Asp33 diads are primarily in the proper orientation of the histidines and in the maintenance of the complicated hydrogen bond network in the active site. Similar roles were proposed for aspartates in the triads of serine proteases (Markley and Westler, 1996; Steitz and Shulman, 1982) and, more recently, for aspartate-121 of

RNase A (Quirk and Raines, 1999).

Comparison of the pKa results with the pH-rate profiles of the studied proteins

(Hondal, 1997; Zhao, 1996) suggests that effect of pH on PI-PLC catalysis can not be explained by simple protonation/deprotonation of general acid and general base. Most likely acid-base equilibria in the active site are more complicated and/or significantly influenced by binding of the substrate.

2.3.4 Binding of substrate analogs to PI-PLC as monitored by H HSQC

Absence of good inhibitors of PI-PLC has significantly hampered progress of its structure-function studies and development of PI-PLC-specific drugs (Bruzik, 1997;

Bruzik and Tsai, 1994; Martin and Wagman, 1996; Roberts et al., 1996). The situation is exemplified by the fact that substrate analogs in the crystal structures of both bacterial

47 (Heinz et al., 1995) and mammalian (Essen et al., 1996) enzymes lack diacylglycerol, an important portion, both structurally and functionally, of the their substrate.

NMR spectroscopy is widely used to investigate protein-ligand interactions

(Byeon et al., 1993; Harlan et al., 1994) due to its ability to monitor conformations of both polymers and small molecules in solution. Furthermore, heteronuclear NMR enables one to use high concentrations of ligands thus allowing to study relatively weakly bound species.

In our early efforts in the NMR studies of bacterial PI-PLC we have resorted to slower isomer of the phosphorothioate substrate analog, Sp-DHPsI. This compound is turned over more than 10^ times slower than the regular substrate ((Hondal et al.,

1997b);see also Chapter 4), but the rate is still too high for NMR experiments with the

WT. Therefore, active site mutant, R69D, was used in experiments with this substrate analog.

Without total NMR assignment of the backbone amide signals that region of the

‘"N-'H HSQC spectrum is not very informative about changes occurring in the protein.

On the other hand, arginine region of the spectrum has only nine well-resolved signals, corresponding to ^NH o f nine arginines in the WT’s sequence (Figure 2.13, A). Partial assignment of the arginine signals was made by site-directed mutagenesis. Arginine side chains in PI-PLC’s tertiary structure are localized in the active site (Arg69 and Argl63), protein’s interior (Arg64 and Arg71) and surface (the other five) (Heinz et al., 1995), thus providing an excellent tool to monitor conformational changes in the enzyme.

48 o D

-88

-84

-86

-88

ppm

15, -84 B -86 -88

ppm

-84

A r g 6 9

A r g 1 6 3

A r g 7 1 -88

ppm ppm

Figure 2.13 : Arginine side chain (®NH) region of HSQC spectra of uniformly labeled PI-PLC: (A) WT; (B) R69D; (C) R69D + 8 mM Sp-DHPsI; (D) WT + 10 mM 2- MeO-DHPI. All samples were in 50 mM HEPES, pH 7.5, protein concentration was ca. 0.4 mM. Spectra were acquired on Bruker DRX-800 spectrometer at 37°C. 4 9 105

-110

-115

4# 120 «N LA O -125

■ • • -130

135

ppm T l I t - I I I ■ t "T—f—I—!~ r t - -rr -i i- r i i i -t ■ ppm 11 10 'H

Figure 2.14: Backbone amide region of '^N-'ll IISQC spccJnim of the uniformly '*N-labeled RfiVl) IM-Pl.C in the presence of 8 mM Sp-l)Msl'I acquired at 800 MHz at 37“C. Sample is in 90% HiO/lO'Mi DiO, 50 mM HFPES, pH 7.5. Spectrum of free R69D has two arginine signals missing (Figure 2.13, B); one from Arg69 and second from its neighbor, Arg71. Additionally, signal of Agrl63 is shifted slightly upfield. All these changes are consistent with electrostatic and some dynamic changes in and around the active site. Upon addition of 8 mM Sp-DHPsI (Figure

2.13, C), signal o f Arg71 reappears in somewhat different position. Meanwhile, Argl63’s signal is moved almost three ppm units downfield. Small residual signal at the original position of Argl63 indicates that bound and unbound protein species are in slow exchange on the NMR time scale.

Spectra of the backbone amide region of free R69D mutant (Figure 2.14) and

R69D in the presence of 8.0 mM Sp-DHPsI (Figure 2.15) have changes analogous to those observed in the arginine region. The differences between the two spectra, which are spread over all spectral region, include shifts in signals’ positions as well as changes in signals’ intensities, both positive and negative. Although a detailed interpretation of the above changes is not possible at this time, they are a clear indication of a significant disturbance in the protein conformation upon ligand binding.

2-deoxy-PI was reported to be an inhibitor of PI-PLC (Martin and Wagman,

1996). When we attempted to use 2-deoxy-DHPI as a ligand in binding studies with WT enzyme, we have foimd that it is a poor substrate with the turnover rate similar to that of

Sp-DHPsI (data not shown). Major products of its cleavage by the enzyme are 1,6-cyclic phosphate 2 -deoxyinositol, phosphatidic acid, and 1-phosphate 2-deoxyinositol. It appears that a water molecule is able to bind in the space normally occupied by 2-OH group and attack the phosphate. Also, it is likely that replacement of 2-OH with a

51 hydrogen atom changes conformation of the inositol ring in such a way that

intramolecular reaction with 6 -OH becomes more favorable.

The third ligand used in oiu* investigation was 2-methoxy-DHPI, which is significantly more stable than the previous two, allowing studies with the WT enzyme.

Addition of 10 mM 2-methoxy-DHPI to the uniformly '^-labeled WT PI-PLC produced much less dramatic changes in the spectra than those observed in case o f R69D and Sp-

DHPsI (Figure 2.13, D). In the arginine region, only signal from Arg69 has shifted downfield, and there is no evidence for the slow exchange between bound and unbound species of the enzyme. Likewise, backbone amide region of the spectrum (not shown) has only a handful of shifted signals in a sharp contrast to the results with R69D and Sp-

DHPsI (Figure 2.15).

On the other hand, addition of 2-methoxy-DHPI to the '^-labeled R69D mutant produced very similar changes to those coursed by addition of Sp-DHPsI (data not shown). However, judging from the intensity of the residual peaks of the unbound protein, R69D binds 2-methoxy-DHPI much weaker than Sp-DHPsI. This observation is consistent with the fact that modifications of the inositol’s 2-OH, as a group, produce less potent inhibitors than most other alterations of the PI-PLC’s substrate (Martin and

Wagman, 1996; Roberts et al., 1996; Ryan et al., 1996).

In summary, NMR binding studies with the available substrate analogs of PI-PLC could not unambiguously resolve the question about possible protein’s conformational change upon substrate binding. It is possible that conformational changes observed upon analogs’ binding to the R69D mutant are caused by this active site mutation. On the other hand, we can not rule out the possibility that WT enzyme is just less susceptible to the

52 conformational changes, and a weak inhibitor (such as 2-methoxy-DHPI) can not give rise to those. This assumption is supported by the fact that binding of this analog did not affect signal of Argl63 (Figure 2.13), which is an important part of the active site’s recognition of the inositol moiety (Heinz et al., 1995).

53 CHAPTER 3

DESIGN AND CHARACTERIZATION

OF A CATALYTIC METAL BINDING SITE

3.1 INTRODUCTION

Bacterial and mammalian PI-PLCs differ not only in their structure, but also in

their substrate specificity and product profile (i.e., IcPrIP ratio) (Bruzik and Tsai, 1994).

While substrate specificity can be easily rationalized from structural data (Essen et al.,

1996; Heinz et al., 1995), the reasons why mammalian enzymes are able to produce IcP and IP simultaneously, while bacterial enzymes’ reaction is clearly sequential, are poorly understood (Heinz et al., 1998).

As it was mentioned already in the Chapter 1, the most significant difference in the structure of the active site between mammalian and bacterial PI-PLC is the presence of a calcium ion in the active site of the mammalian enzyme (Figures 1.5). Bacterial enzyme has a guanidine group of arginine-69 in essentially the same position (Figure

1.6 ). Similar positioning of these two positively charged moieties suggests that they carry out the same or very similar fimction(s) in the catalysis. This notion poses several questions:

54 what exactly that function is, if it can be successfully accomplished by two groups as

different as metal ion and guanidine group?

are other changes in the active site (in particular, positions of the homologous

histidines, histidine-314 and histidine-32) related to the metal-to-guanidine switch?

One of the possible ways to clarify those questions is to attempt a conversion of metal- dependent enzyme into metal-independent one or vice versa. In the bacterial PI-PLC side chain of Arg-69 is hydrogen-bonded by aspartate-33, aspartate-67, and glutamate-117, which creates a rough blueprint for the future metal-binding site. It is also necessary to note that positions of those amino acids are very similar to ones in the coordination sphere of the calcium ion in the mammalian enzyme (asparagine-312, glutamate-341 and glutamate-390) (Essen et al., 1996; Essen et al., 1997a).

Metal binding site design in proteins has been employed in many different systems to address variety of questions (reviewed in (Lu and Valentine, 1997; Regan,

1993; Regan, 1995)): to study protein-protein interactions and topology of transmembrane domains (He et al., 1995a; He et al., 1995b), to regulate activity and specificity of enzymes (Higaki et al., 1990; McGrath et al., 1993), and to modify redox chemistry of an enzymatic reaction (Wilcox et al., 1998). Surprisingly, the interchange between positively charged side chains and metal ions was successively achieved only in the metal to amino acid direction (Casareno et al., 1995). To the best of our knowledge, there are no examples in the literature describing a de novo metal site that would replace, completely or partially, function of a lysine or an arginine.

This Chapter describes design and characterization of the metal-dependent variants of the bacterial PI-PLC, which are viewed as tools to address the observed

55 differences in catalysis between mammalian (metal dependent) and bacterial (metal independent) enzymes.

3.2 MATERIALS AND METHODS

Phosphatidylinositol (PI) from bovine brain and l,2-diheptanoyl-sn-glycero-3- phosphocholine (DHPC) were from Avanti Polar Lipids. L-[myo-inositol-2^H]- phosphatidylinositol ([^H]-PI) was from Dupont NEN. 5,5'-Dithiobis[2-nitrobenzoic acid]

(DTNB) was purchased from Aldrich. Oligonucleotides were custom-synthesized at

Integrated DNA Technologies.

3.2.1 Mutants

All mutants were made by “QuickChange” procedure (Stratagene) according to manufacture’s manual.

The following mutagenesis oligos were used (codon of interest is in italics and modified nucleotides are in bold): R69D (underlined is recognition site of Clal (BspDI) endonuclease) - 5 -CGCATTTTTGATATCSiTOGACGTTTAACAGATG-3

5 -CATCTGTTAAACGTCC/i TCGATATCAAAAATGCG-3D67N (with R69D):

5 -GGAGCTCGCATTTTT/U TATTGATGGACG-3',

5 -CGTCC ATCAAT/17TAAAAATGCGAGCTCC-3

D67E (with R69D): 5 -GGAGCTCGCATTTTTGÆ4ATTGATGGACG-3

5 -CGTCCATC AAT rrCAAAAATGCGAGCTCC-3

E117Q: 5 -CAATTATTATGTCTTTAAAAAAACWGTATGAGGATATG-3%

56 5 -CATATCCTCATACrCTTTTTTTAAAGACATAATAATTG-3

R69N (underlined is recognition site of EcoRV endonuclease):

5 '-CGCATTTTTGATATC/L4 CGG ACGTTT AAC AGATG-3

5'-CATCTGTTAAACGTCCGr7TGATATCAAAAATGCG-3':

R69E: 5 -CGCATTTTTGATATAG4CGGACGTTTAACAGATG-3'

5 -CATCTGTTAAACGTCCCrCTATATCAAAAATGCG-3

D3 3 E : 5 -C AATTCC AGG AAC AC ACGÆ4 AGTGGGACG-3 %

5 -CGTCCCACTTrCGTGTGTTCCTGGAATTG-3All mutations were verified by

DNA sequencing of the mutation site(s) and the whole gene. All mutant proteins were purified as described in Chapter 2, but all buffers, except for the final dialysis, included 1 mM EOT A to prevent metal contamination.

3.2.2 Radioactive activity assay of PI-PLC with f H]-PI substrate

The specific activities of mutants were measured according to the procedure reported earlier (Hondal, 1997; Volwerk et al., 1989a) with a few modifications. ^H-PI was mixed with the unlabeled PI from bovine brain to obtain an overall PI concentration of 10 mM and a specific activity of ca. 1.25 x 10^ cpm/mol. Detergent DHPC was also added to the final concentration of 40 mM. The reaction mixture contained 20 faL of this substrate solution and 40 pL of 0.1 M HEPES, pH 7.5. An aliquot of 20 pL of PI-PLC was added to the reaction mixture and incubated at 37°C for 10 min. Reaction was stopped by the addition of 0.5 ml CHCI 3-CH3OH-HCI (66:33:1, v/v) mixture. Phases were separated by a brief centrifugation and radioactivity of 50-100 pi of the aqueous phase was measured by scintillation counter (Beckman). The concentrations of WT PI-

57 PLC and mutants were adjusted so that substrate conversion does not exceed 10-30%.

Enzymatic activity was expressed in pmolxmin'^x(mg of protein)*' or U/(mg of proteins).

Metal ions of appropriate concentrations were added with the enzyme solution.

Activity results did not change if metals were added to the substrate before the addition of the enzyme. 1 mM EDTA was added to the reaction solution to measure enzyme’s background activity (i.e., one without any metal ions present).

pH-activity profile. Activity of R69D over pH range 4.5 - 9.0 (with and without metal ion present) was measured in a buffer containing 50 mM of each acetic acid, MES, and HEPES. pH of that buffer was adjusted by NaOH. pH was measured by the combined pH-electrode using digital pH/ion meter (Orion Research).

3.2.3 R69D NMR titration by Ca~^

Uniformly '^-labeled R69D PI-PLC was purified and its '^ -'H HSQC spectra were acquired as described in Chapter 2. Spectra were taken of the free enzyme and of the enzyme in the presence of CaCh in the concentrations of 0.25 mM, 1 mM, and 5 mM.

3.3 RESULTS AND DISCUSSION

The most straightforward approach in the design of a metal binding site replacing a lysine or an arginine will be a substitution of the positively charged side chain with the carboxylic acid. Therefore, arginine-69 was mutated to aspartate and glutamate with the intention to get the simplest design possible. The expectations were that if metals would

58 activate any of these single mutants, further adjustments could be made in attempt to

improve metal binding properties of the site, its specificity and catalytic efficiency.

R69E mutant is extremely inactive (4x10'^ U/mg, compared to 2200 U/mg for

WT enzyme) and is not activated by any divalent metal ions (data not shown). R69D

mutant, on the other hand, is activated by various metal ions as described in detail below.

3.3.1 Metal activation and metal specificity o f R69D mutant

First of all, activation of R69D mutant by earth-alkaline (main subgroup of the

second group in the periodic table) metals was studied. This mutant is activated to

different extent by Mg^^, Ca^% and Sr^^ (Figure 3.1). Presence of another earth-alkaline

metal, barium, neither activates nor inhibits R69D PI-PLC in the concentrations up to 1

mM (data not shown). Activation data by magnesium, calcium, and strontium are

summarized in Table 3.1.

Metal Vma.x, pmol/(min mg) Metal’s Kd'"", mM

— 0 . 10±0 .0 2 NA

Mg'* 1.73±0.16 0.31 ±0.09

Ca'" 1.61 ±0.08 0.15±0.03

Sr'" 0.71±0.08 0.11 ±0.05

Table 3.1 Kinetic parameters of R69D PI-PLC with earth-alkaline metals.

59 e n

0.0 0 200 400 600 800 1000 nM

Figure 3.1 : Activation of R69D PI-PLC by earth-alkaline metals: magnesium (O), calcium (•), and strontium (▼ ). Solid lines represent non-linear fit of each data set to the Michaelis-Menten equation.).

0 8

0 7

0.6

E I ^ 0.3

0.2

0 0 0 200 400600 800 1000

Figure 3.2: Activation of R69D PI-PLC by transition vc metals: cobalt II ( • ) and manganese II ( ▲).

60 Overall activation by these metals is relatively modest, with difference between

R69D without any metal ions present and maximally activated mutant being only 16-17 fold. Vmax for strontium is approximately one-half of that for magnesium and calcium, both of which provide essentially the same activation to R69D. Effective dissociation constants o f the three metals follow the reversed trend for their ionic radii (Mg"^ ( 0 .6 6 À)

> Ca-^ (0.99 Â) > Sr^" (1.12 Â) (Lide et al., 1996)).

Out of other divalent metals tried, only cobalt (II) and manganese (U) has shown some activation (Figure 3.2). In contrast to the earth-alkaline metal ions, these transition metals increase activity of R69D at low concentrations, reaching maximum at 50-100 fiM. Maximum activation is less than 5-fold for manganese and about 7-fold for cobalt.

Further increase in metal concentration inhibits enzyme’s activity, but it stays above the background level even at 1 mM ion concentration for both metals.

Other metal ions either do not have effect on the R69D’s activity (like Cd~") or inhibit its activity (e.g., Zn“" and La^ ). Presence of the aspartate residue in the position

69 is essential for the formation of the metal binding site since other mutants in this position (R69A, R69G, and R69C) are not activated by metal ions. Besides, unlike R69G and R69A mutants (see Chapter 4), R69D PI-PLC is not activated by guanidine chloride.

pH-activity profile of R69D PI-PLC (Figure 3.3) is significantly narrower than that of the WT enzyme (Hondal, 1997), with half-maxima at pH 6.4 and 8.2 at the ascending and descending limbs, respectively. In the WT enzyme the ascending limb is shifted about 2 pH units to more acidic values, while the descending one is very similar to R69D’s. Presence o f the calcium ion does not have any effect on the descending limb of R69D enzyme, but shifts the ascending one by about 0.5 pH units to higher values.

61 120

100

SS 80

> m 60 Î « 40

20

O 5 6 7 8 9 pH

Figure 3.3: pH-activity profile of R69D PI-PLC without any metal present (solid line, dark circles) and with 0.8 mM CaClz (dashed line, open circles). Activities are normalized relative to the maximum in each case. At their respective maxima, activity with calcium is 15-fold higher than that of the apoenzyme.

Bacterial WT PI-PLC is inhibited, albeit to different extent, by almost all divalent

metal ions (Taguchi et al., 1980). Mechanism of such inhibition is thought to be by non­

specific binding of the ions in or near the active site and/or by ions’ interactions with the

negatively charged substrate. Therefore, observed activation of R69D PI-PLC by various

metal ions is a specific property of this mutant, resulting fi-om the metal ion’s binding in

the active site of the enzyme.

Apparent dissociation constants in sub-millimolar range for all three earth-

alkaline metals (Table 3.1) should be considered very successful for a de novo metal

binding site (McPhalen el a!., 1991). For a comparison, affinity to calcium in the active site of the mammalian PI-PLC 81 is only about ten-fold higher (Wu et ai., 1997). 62 Quite surprising feature of the engineered binding site is its low discrimination

between magnesium and calcium ions: they activate the protein to the same extent and

affinity for calcium is only slightly higher. Usually, metalloenzymes utilizing calcium or

magnesium are only active with one but not the other (Cowan, 1998; McPhalen et al.,

1991). In fact, ribozymes in presence o f calcium are able to fold and bind its substrate,

but no cleavage can be observed (McConnell et al., 1997). The promiscuity o f the

introduced metal binding site can be explained by its crude design. Consequently, it could

be viewed as a model to study specificity and evolution of metal-binding sites.

Effect of the bound calcium on the R69D’s pH profile (Figure 3.3) can not be

explained by an effect of the positive charge on pKa of a histidine since imidazole’s pKa

should decrease, not increase, under those conditions. Besides, due to expected central position of the metal ion one would expect similar effect on both general acid (His82) and general base (His32). These results emphasize one more time the complex nature of the pH-rate profiles of PI-PLC (see also Chapter 2).

The most obvious explanation for the low catalytic efficiency of the metal ions would be an improper positioning of the latter in the active site. Further studies were undertaken in attempt to establish coordination sphere and position of the metal ion in the active site and its effect on the structure and dynamics of R69D PI-PLC.

3.3.2 Effect o f calcium binding on R69D structure and dynamics

As has been discussed in Chapter 2, mutation of arginine-69 to aspartate brings significant changes to the active site and its proximity as monitored by heteronuclear

NMR spectroscopy (Figure 3.4, A and B; also, compare Figures 2.6 and 2.14). Those

63 D 86 o

88

ppm

84

88

ppm 15,N B c 0

-

ppm

84 Arg69 ^ o o 86 Arg163 Arg71

ppm ■—I--- r---1--- 1--- 1--- ^ ppm 8 "H

Figure 3.4: Arginine side chain (®NH) region of HSQC spectra of uniformly labeled PI-PLC: (A) WT; (B) R69D; (C) R69D + 1 mM CaCb; (D) WT + 5 mM CaCL. All samples were in 50 mM HEPES, pH 7.5, protein concentration was ca. 0.4 mM. Spectra were acquired on Bruker DRX-800 spectrometer at 37°C.

64 Ob o <6 -105

110

115

120 %. 15iN s 125

130

135

ppm ■1 I ■ T-T—r - f - f T- l ■ I T l ' I - -T-T—I—I— T-T—r—T-1—r—1—r-T-T—T—I—r—r-T—i i i i w i i » ppm 11 10 'H

Figure 3.5: Backbone amide region of '*N- 'il HSQC spectrum of the uniformly "'N-labeled R69D IM-Pl.C in the presence of 5 mM CaCi, acquired at 800 MHz at 37°('. Sample is in 90% HiO/10% l),0, 50 mM HI:I»I-S, pH 7.5, changes are consistent with localized conformational changes, perturbed dynamic

properties, and charge redistribution in the active site, as would be expected from such

non-conservative substitution. Specifically, in the arginine region of HSQC

spectrum upon R69D mutation signals from Arg69 and Arg71 disappear, while signal of

Argl63 is shifted slightly up field.

Upon addition of calcium ion to the R69D PI-PLC (Figure 3.4, C and D), signal

from Arg71 gradually comes back at the same position as in WT enzyme, while signal of

Argl63 shifts downfield close to its original position in the WT. Thus, introduction of the

calcium ion into the active site of R69D mutant is equivalent to the presence of guanidine

group of Arg69 in the WT enzyme.

Addition of calcium also induces alterations in signal positions and intensities in

the backbone amide region of the HSQC spectrum after (Figure 3.5; compare with Figure

2.14). Unfortunately, these changes can not be rationalized without the complete NMR

assignment, but they are consistent with the idea that binding of calcium ion induces

localized subtle perturbations to the structural and dynamic properties of the protein.

3.3.3 Characterization o f the metal binding site

NMR titration of R69D PI-PLC with calcium indicates that metal ion in this mutant is located in the position occupied by the guanidine group in WT enzyme, therefore its coordination sphere should include Asp33, Asp67, Asp69, and Glul 17

(Figure 1.6). Series of single and double mutants (on top of the R69D mutation) in those positions were constructed in order to verify participation of these amino acids in the metal coordination and in attempt to alter metal-binding properties of the site.

66 First, all four carboxylic acids were mutated (one at a time) to the corresponding

amide thus creating the following mutants; D33N/R69D, D67N/R69D, R69N, and

R69D/E117Q. Activity of the latter three proteins was not changed relative to R69D

mutant without metal ions, but all three lost their ability for metal activation. Entirely

different story was observed for D33N/R69D double mutant.

Activity of the D33N/R69D PI-PLC is lower by almost 70-fold compared to the

R69D single mutant. Its activation by magnesium and calcium has well-pronounced sigmoidal form (Figure 3.5) with Hill’s coefficient from non-linear fit being 2.4 and 3.4, respectively. D33N/R69D mutant is activated by magnesium 500-fold as compared to about 100-fold activation by calcium. Furthermore, apparent dissociation constant of the magnesium ion is almost three-fold lower than calcium’s one.

Additionally, metal binding site was probed by D67E/R69D double mutant and

D33N/D67E/R69D triple mutant. D67E/R69D PI-PLC has activity similar to the R69D single mutant and is not activated by divalent ions. On the other hand, the triple mutant’s activity is the same as that of D33N/R69D double mutant, but 1 mM concentrations of magnesium and calcium activated it 40- and 10-fold, respectively. Further characterization of the triple mutant was not undertaken since its activation parameters were worse than those of the D33N/R69D enzyme.

Mutagenesis analysis of the putative metal binding site has confirmed that metal coordination is carried out by the side chains of Asp33, Asp67, Asp69, and Glul 17.

Presumably, the phosphate group of the substrate complements the coordination sphere.

Quite unexpectedly, D33N/R69D double mutant shows an altered dependence of the enzyme’s activity on the metal concentration for both magnesium and calcium

67 OS

oi

0 0 0 500 1000 1500 2000 [Me"*], nM

Figure 3.6: Activation of D33N/R69D PI-PLC by magnesium (•) and calcium (O). Solid lines are results of non-linear fit to the Hill’s equation with the following parameters: Mg~" - Vmax = (0.72±0.09) U/mg, IQ = (0.37±0.06) mM, and n = 2.4±0.5; Ca'" - V^ax = (0.130+0.007) U/mg, IQ = (0.83±0.04) mM, and n — 3.4±0.5.

(Figure 3.6). It is unlikely that aspartate to asparagine mutation has introduced additional metal binding site(s) into the protein, therefore sigmoidal shape of the dependence has some other explanation(s). It seems the most feasible rationalization would be the following: D33N/R69D PI-PLC can exist in two conformations, one active, another inactive. Binding of the metal ion in the active site shifts the equilibrium to the active conformation. The conformational change do not need to be dramatic, repositioning of one or two amino acids in the active site should be able to account for the observed effects.

68 No matter what are the reasons behind the sigmoidal activation, D33N/R69D

mutant is able to discriminate between magnesium and calcium much better than R69D

PI-PLC. Both total activation (by 5-fold) and dissociation constant (by 3 fold) favor

magnesium ion. Unfortunately, this discrimination comes at a price of lower maximal

activity and lower affinity for the metal ion. It appears that changes of the coordinating

carboxylic acids is not going to be effective in fine tuning of the metal affinity and

specificity. Therefore, some other, more subtle, manipulations are needed for that

purpose.

3.3.4 Low catalytic efficiency of the metal ions: possible explanations,

implications and new directions.

Even though newly introduced metal binding site has a very good affinity for the

metal ions and its location coincides with the position o f the guanidine group in the WT

enzyme, metal ions fail to activate R69D mutant to anywhere near the WT. In

comparison, mammalian PI-PLCs are activated by calcium more than 1000-fold (Allen et

al., 1997; Grobler and Hurley, 1998; Wu et al., 1997; Zhou et al., 1999). Two

explanations seem to be the most plausible: (i) positioning of the metal ion is still not

optimal - for example, it does not coordinate both phosphate and 2-OH of inositol, as

was proposed for the PI-PLC 51 (Essen et al., 1996); (ii) switch from guanidine to a

metal ion requires additional reorganization of the catalytic residues, corresponding, for

instance, to the positions of homologous histidines in bacterial and mammalian enzymes

(Heinz e/a/., 1998).

69 One of the possible approaches to clarify those issues would be a comparative

study of the R69D mutant (and its variants) of the B. thuringiensis enzyme with the

recently discovered small calcium-dependent PI-PLC from Streptomyces amibioticus

(Iwasaki et al., 1998). The latter enzyme is of similar size and has the same substrate preference as the B. thuringiensis enzyme, which makes the comparison of the two much more straightforward than that of the bacterial and mammalian enzymes.

Interestingly, amoimt of R69D activation by the metal ions is very similar to the activation of R69C mutant by modifications carrying amino group (Chapter 4).

Therefore, these results might be indicative of the amount of the electrophilic catalysis in the mechanism of PI-PLC. The estimated 1.5 kcal/mol attributed to the stabilization of the T.S. by a positive charge is consistent with the little negative charge development in the transition state of the reaction as elucidated by LFER studies (Chapter 5).

70 CHAPTER 4

CATALYTIC ROLE OF ARGININE 69 IN B.THURINGIENSIS PI-PLC:

SITE-DIRECTED CHEMICAL MODIFICATIONS AND THIO-EFFECTS

4.1 INTRODUCTION

4.J.I Phosphorothioates as mechanistic probes o f enzymatic reactions

Phosphorothioates, analogs of phosphate with one of non-bridging oxygens

replaced by a sulfur atom, were one of the earliest (Eckstein et al., 1982; Frey, 1989) and still are one of the most popular mechanistic probes of the phosphoryl transfer reactions,

for both enzymes (Hollfelder and Herschlag, 1995) and ribozymes (Herschlag et al.,

1991). Particularly valuable phosphorothioates have proved to be for steric course elucidation of phosphoryl transfer reactions (Bryant et al., 1981; Pliura et al., 1980;

Rosario-Jansen et al., 1988; Tsai et al., 1995). However, mechanistic interpretations of the kinetic effects of the resulting oxygen to sulfur substitutions (non-bridging thio- effects) are still stirring up some controversy even for well-studied RNase A (Breslow and Chapman, 1996; Herschlag, 1994).

71 Chemical reactivity of phosphorothioate diesters does not significantly differ from that of the corresponding phosphates: it is about 2-4 fold for intermolecular and even less so for intramolecular reactions (Herschlag et al., 1991). Therefore, observed thio-effects

(which are predominantly >1) almost entirely result from either disruption of favorable protein-substrate interactions or from newly introduced unfavorable ones. In the former case, the interactions being affected are hydrogen bonding and/or a metal complexation, since sulfur and oxygen differ considerably in their ability to form hydrogen bonds and in their preference for the metal partners (Frey, 1989).

PI-PLC RNase A

RCOO

RCOO

HB-Arg69 OH

OH HO OH

HB-His119

Figure 4.1: Bacterial PI-PLC and RNase A: a mechanistic analogy. Shown are structures of both substrates and major components of enzymatic catalytic mechanism: general base (His-32 and His-12), general acid (His-82 and His-119), and phosphate activation (Arg- 69 and Lys-41). pro-S and pro-R oxygens of both prochiral phosphate groups are labeled with S and R, respectively.

72 4.1.2 Thio-effects o f RNase A and PI-PLC: lysine vs. arginine

Bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) displays unusually high non-bridging thio-effects, which are associated with the catalytic function of arginine-69 (Hondal et al., 1997b). While PI-PLC and RNase A have significant similarities in both structures of their respective substrates and mechanisms employed in the catalysis (Figure 4.1), thio-effects are significantly different for these two enzymes:

Rp isomers have thio-effects of 2 and 40, and Sp isomers - 70 and over 100,000 for

RNase A and PI-PLC, respectively (Burgers and Eckstein, 1979; Hondal et al., 1997b).

Such tremendous difference in thio-effects suggests fundamentally different interactions of the two enzymes with the phosphate groups of their respective substrates. The only apparent distinction in the phosphate activation between these two enzymes is that in

RNase A activation of the phosphate group was assigned to a lysine - Lys-41 (Blackburn and Moore, 1982; Messmore et al., 1995), while in PI-PLC similar role was suggested for an arginine (Arg-69) (Gassier et al., 1997; Heinz et al., 1995; Hondal et al., 1997b).

Replacement of PI-PLC’s arginine-69 with lysine had dramatic effects on both activity and thio-effects (Hondal, 1997; Hondal et al., 1997b), but since structures of these amino acids are very different, it was not clear whether side chain’s length, or the structure of the terminal functional group, or both were responsible for the observed results.

4.1.2 Chemical modification o f cysteine: extension of the genetic code

While Mother Nature successively operates with the limited set of only 20 amino acids, quite often protein chemists and enzymologists in their studies require properties of

73 the side chains, which are not available naturally. Several approaches have been

developed to remedy the situation.

The earliest method was chemical modification by residue-specific reagents

(reviewed in (Came, 1994)). This technique, widely used in the early enzymatic studies, is very non-specific, modifying all accessible side chains with similar reactivity. Only in very few cases it was possible to modify specifically an amino acid, typically in the active sites of enzymes, with significantly improved reactivity. Probably the highest specificity overall for this method was achieved for cysteines (Kaiser and Lawrence,

1984), due to the facts that this amino acid is rarely solvent-accessible and it is the best nucleophile among natural amino acid.

Chemical synthesis can be used to introduce almost any side chain structure

(provided a suitable protecting group can be found) into peptides, but it is only practical for proteins and peptides of small size (ca. <100 amino acids) (Wilken and Kent, 1998).

The most elaborate method for the introduction of unnatural amino acids into proteins, developed by Peter Schultz and coworkers (Anthony-Cahill et al., 1989; Norea et al., 1989), takes advantage of in vitro translation system and tRNA chemically charged with the structure of interest. While this approach is applicable to wide variety of protein

- amino acid combinations, technical difficulties and low yields have limited its distribution.

Site-directed chemical modification (sometimes called “chemical elaboration of cysteine”) successfully combines powers of site-directed mutagenesis and chemical modification of cysteine side chain (Figure 4.2). Since first introduction by F. C. Hartman

74 ciç f— Cys .s B \ „ I \ / \ S R"

Figure 4.2: Reactions typically used for the chemical modification of cysteine: modification reagents either have a good leaving group (such as iodine, bromine, or chlorine, with formation of C-S bond (A), or an S-S bond, resulting in the formation of a disulfide bond in the modified side chain (B).

in his work on ribulosebisphosphate carboxylase/oxygenase (Smith and Hartman, 1988), this technique has been used in the numerous systems: in modulating properties of subtilisin (DeSantis et al., 1998), as a probe for ion-channel properties (Foong el al.,

1997), for site-directed incorporation o f spin-labels (Hubbell et al., 1996; Lin et al.,

1998), for investigation of membrane-spanning proteins (Chen et al., 1997), and for improving catalytic power of natural enzymes (Earnhardt et al., 1999). Particularly valuable this approach has been proven to be for the systematic studies of arginines and lysines in enzymatic catalysis and substrate/ligand binding (Dhalla et al., 1994; Gloss and

Kirsch, 1995; Messmore et al., 1995; Paetzel et al., 1997).

Bacterial PI-PLC is a very suitable target for the site-directed chemical modification since WT enzyme does not contain any cysteine residues (Lechner et al.,

1989), thus every introduced cysteine will be a unique target for the modification.

75 This chapter describes application of site-directed mutagenesis and site-directed chemical modification at the position 69 of the bacterial PI-PLC to delineate the origin of the observed non-bridging thio-effects and the enzyme’s preference for the Rp isomer.

4.2 MATERIALS AND METHODS

DPPsI (mixture of Rp and Sp isomers) and enantiomerically pure (2R)-1,2- dioctanoyloxypropane-3-(thiophospho-lD-myo-inositol) (DOsPI) were supplied by Dr.

Karol S. Bruzik (University of Illinois at Chicago).

2-Chloroacetamidine HCl was fi-om Lancaster Synthesis, 2-chloroacetylnitrile and

5,5'-dithiobis[2-nitrobenzoic acid] (DTNB) were from Aldrich, 2-bromoethylamine and

3-brompropylamine were from Sigma. All other reagents were of the highest grade available.

4.2.1 Synthesis o f dithio-bis(acetylamidine) (DTBA)

Combination of two previously published procedures was used for synthesis of

DTBA (Schaeffer and Peters, 1961; Westland et al., 1972). In brief, 7.5 g (6.3 ml; 0.10 mol) of CI-CH2-CN (Sigma) was added dropwise to ice-cold 100 ml 0.1 M CHsONa in methanol. Mixture was stirred @ RT for I hr, then 5.30 g (0.1 mol) of NH4CI was added and stirring continued until all salt was dissolved (ca. 1 hr). The resulting solution had light yellow color and was concentrated on rotavap, remaining solid (12.5 g), chloroacetylamidine ( 1), had dark brown-red color, attributed to impurities. 1 was

76 triturated with dry ether (2x50 ml) to remove any unreacted nitrile. After that, 1 was used in the subsequent synthesis without additional purification. All 1 was dissolved in 50 ml of H 2O, small excess (24.3 g) of Na^SzOs SHiO was added and mixture was incubated @

100°C for 1 hr; black flakes of impurities were filtered out and clear solution was cooled on ice. Golden-brown precipitate (acetylamidine thiosulfate, 2; 13.1 g final) was recrystallyzed from H 2O. All 2 was dissolved in 150 ml of 1 N H 2SO4, and 5.8 g of thiourea was added; mixture was incubated on boiling water bath for 6 hrs. Solid sulfur was filtered out, and solution was lyophilylized. Resulting solid was recrystallyzed successively from 1:1 water-ethanol and fi'om water. Final product, DTBA (3, 1.7 g), slightly yellowish white solid, was used for protein modification without further purification.

4.2.2 Arg69—K2ys mutant

R69C mutant was made using “QuickChange” (Stratagene) procedure using following oligos (changed nucleotides are in bold and 69 *^ codon is in italics): 5'-

CGC ATTTTTGAT AT ArGrOGACGTTT AAC AGATG-3' (sense) and 5'-

C ATCTGTT AAACGTCC/fCWT AT ATC AAAAATGCG-3'(antisense).

The very first batch of R69C mutant was purified with 10 mM EDTA and 1 mM

DTT in all buffers, except for the final dialysis, to prevent possible metal contamination and sulfide oxidation. After it became evident (see below) that cysteine residue is not exposed to solvent, the regular protein purification procedure (Chapter 2) was used.

77 4.2.3 Quantification o f free thiol with DTNB

0.5-1 mg o f unmodified or 2-3 mg of modified R69C mutant were dissolved in 5

M Gnd-HCl (pH 7.5), and DTNB was added to final concentration of 1 mM. Reaction

mixture was incubated at RT for 15 min and concentration of the released pigment was

measured at 412 run using extinction coefficient of 13,600 M'*cm’' (Wright and Viola,

1998).

4.2.4 Chemical modification reactions

~10 mg of PI-PLC R69C was dissolved in buffer containing 50 mM Tris (pH 8.0),

3.5 M guanidine chloride, 5 mM EOT A and 0.2 M (0.1 M in case of DTBA) modifying reagent. Reaction mixture was flushed with argon and incubated in darkness at room temperature for 1-3 hrs. Incubation times were determined individually for each reagent in trial experiments. Degree of modification was monitored by free thiol titration with

DTNB. After incubation, reaction mixture was dialyzed stepwise successively against four buffers:

1) 2 M urea, 100 mM HEPES, 100 mM NaCl (pH 7.0);

2) 1 M lu-ea, 50 mM HEPES, 100 mM NaCl (pH 7.0);

3) 20 mM HEPES, 100 mM NaCl (pH 7.0);

4) 10 mM HEPES (pH 7.0).

Protein precipitate was span down and supernatant concentrated and lyophilized.

Recovery of the soluble protein after this refolding procedure varied in the range of 70-90

%.

78 4.2.5 Native PAGE and isoelectrofocusing

Native PAGE of protein samples were run on Phastsystem (Pharmacia) using homogeneous 20 % gels according to manufacturer’s recommendations.

Isoelectrofocusing (lEF) was conducted on Phastsystem (Pharmacia) in pre­ manufactured pH gradient (pH 4-6.5). Both native PAGE and LEE gels were silver- stained according to the corresponding manufacturer’s protocols.

4.2.6 Mass-spectrometry o f modified proteins

Electrospray ionization mass-spectra of all modified proteins as well as of R69C mutant were done in biological mass-spectroscopy facility at OSU.

4.2.7 P-NMR assays with DPPsI

^'P-NMR assays were done on Bruker’s DRX-500 and DRX-600 NMR spectrometers both equipped with 5 mm broadband probe and temperature control unit.

All reactions were done at 25°C in 50 mM HEPES buffer, pH 7.5. Mixture of Sp and Rp isomers (ca. 6:4 ratio) of DPPsI at final concentration of 10 mM was dispersed into micelles in bath sonicator with four-fold excess of detergent, DHPC. 0.5 ml aliquots of the prepared substrate were used in each reaction. Reactions were initiated by addition of the appropriate amounts of protein, diluted in the same buffer. If enzymatic rates toward the two isomers were significantly different, second aliquot of protein (as a rule, of significantly higher concentration) was added after the reaction with faster (Rp) isomer.

Rates of the reactions were calculated fi"om the linear portions of product (IcPs) formation.

79 4.2.8 Determination o f bridging thio-effects.

Reactions with the chromogenic substrate, DOsPI, were done as reported earlier

(Honda! et al., 1997b; Mihai et al., 1997). Reaction mixtures included 1 mM DOsPI, 5

mM DTNB in 50 mM HEPES, pH 7.5. The initial reaction velocity (Vo) was obtained by

monitoring the change in absorbance at 412 nm. Activities of WT, R69C-AA, and R69C-

EA from this assay were compared to those in the radioactive assay (Chapter 3) to obtain

the bridging thio-effects.

Lys Arg 6.33 A 6.22 A

Cys-EA Cys-AA NH^ 5.40 6.61 A

NH, Cys-PA Cys-TAA NH 6.58 A 7.85 A

Figure 4.3: Comparison between the side chains of arginine, lysine, and modified cysteins. Abbreviations: AA - acetamidine, TAA - thioacetamidine, EA - ethylamine, PA - propylamine.

80 4.3 RESULTS AND DISCUSSION

4.3.1 Chemical modification of R69C mutant and characterization of the modified proteins.

Wild-type (WT) PI-PLC does not contain any cysteines, so mutation of arginine-

69 to cysteine (R69C) created a unique target for the chemical modification. Total four modifications has been made; two “arginine analogs” (cysteine-acetamidine and cysteine- thioacetamidine) and two “lysine analogs” (cysteine-ethylamine and cysteine- p ropy lamine). Resulting side chains are compared in Figure 4.3 to arginine (as in WT) and lysine (as in R69K mutant).

Even though arginine-69 is located in the active site, cysteine side chain in R69C mutant appears to be completely shielded from the solvent. Under native conditions, neither modification reagents, nor Ellman’s reagent are able to react with the thiol in the pH range 7.0-9.0. Therefore, modifications were made imder partially denaturing conditions in the presence of 3.5 M guanidine chloride. Modified proteins were subsequently refolded by stepwise dialysis against decreasing concentrations of chaotropic agents.

Titration of the modified proteins with DTNB confirmed that reactions were quantitative with the residual content of the fi-ee thiol in all cases being less than 5%.

Analysis of modified proteins by electrospray mass spectrometry confirmed specificity and stoichiometry of the modifications (Table 4.1). Purity of the modified enzymes was checked by native-PAGE and in all cases only one band was visible (Figure 4.4, A). 81 Isoelectric focusing demonstrated that modifications of R69C restored its pi to the levels of WT and R69K mutant (Figure 4.4, B).

Found MW, Protein Calculated MW, Da Da

R69C 34,459 34,361

R69C-EA 34,503 34,510

R69C-PA 34,517 34,522

R69C-AA 34,516 34,521

R69C-TAA 34,548 34,552

Table 4.1 Electrospray ionization mass-spectrometry analysis of PI-PLC R69C mutant and its modifications.

B

1 2 3 4 5 6 7

1 2 3 4 5 6 7

Figure 4.4: (A) lEF (pH 4.0 - 6.5) and (B) native-PAGE (20%) gels of PI-PLC. Lanes: 1 - WT, 2 - R69C-AA, 3 - R69C-TAA, 4 - R69C, 5 - R69C-PA, 6 - R69C-EA, 7 - R69K.

82 Specific activity, pmol/(min mg) Enzyme DPPI’ Rp-DPPsi” Sp-DPPsl” WT 2200 53 .007

R69C-AA 160 4.0 .001

R69C-TAA 50 1.3 .00043

R69C-PA 1.5 .043 .0038

R69C-EA .50 .014 .0046

R69K .10 .0029 .0037

- radioactive assay; - NMR assay.

Table 4.2. Specific activities of PI-PLC with different side-chains at position 69 toward oxygen and phosphorothioate substrates.

4.3.2 Effect of the side chain structure on activity and thio-effects

Activities of modified proteins (along with WT and R69K mutant) toward oxygen

(DPPI) and toward two isomers of sulfur (DPPsI) substrates are summarized in Table 4.2.

First of all, activities toward Sp-isomer of the thiophosphate substrate are remarkably similar by all proteins, the difference being less then twenty fold between the highest

(WT) and the lowest (R69C-TAA) activities. The unexpectedly low activity of the R69C-

TAA protein can be attributed to the unfavorable steric effects originating fi'om the

83 significantly larger size of this side chain (Figure 4.3). Probably, those effects are also

lowering activities of this protein toward both Rp isomer of the sulfur and oxygen substrates. This notion has a strong support from the analysis of thio-effects and stereoselectivity (see below). In any case, these possible steric effects, if present, appear to be small relatively to the catalytic effect from other factors under consideration.

In contrast to Sp-DPPsI, activities toward oxygen substrate and Rp-DPPsI show differences of up to twenty thousand fold and well-defined patterns as analyzed below.

First, activities toward Rp-DPPsI go in parallel with those toward DPPI, and, as a result, thio-effects o f Rp isomer (ko/kap) for all analyzed proteins is essentially the same (ca. 40,

Table 4.3). These results suggest that Rp thio-effects are not caused by phosphate’s interaction (or by alteration of thereof) with side chains at the position 69, but is coming from changed intrinsic reactivity of the thio-substrate and/or from an unfavorable interaction of the sulfur atom with another part of the active site. Second, as a group, enzymes with a monodentate side chains (i.e. “lysine series” - R69K, R69C-EA, and

R69C-PA) have substantially lower activity than enzymes with a bidentate functional group (i.e. “arginine series” - WT, R69C-AA, and R69C-TAA). This difference is even more pronounced in the thio-effects of Sp-DPPsI (ko/kgp is higher by at least three orders of magnitude for the members of “arginine series”) and stereoselectivity. Even R69C-

TAA, which has the lowest activity within “arginine series”, has both Sp thio-effect and the stereoselectivity indistinguishable from WT and R69C-AA (Table 4.3). Therefore, possible steric effects for this protein, discussed earlier, do not interfere with the complete picture of catalysis.

84 Interestingly, stereoselectivity of the best enzyme in the “lysine series”, R69C-

PA, is very similar to the stereoselectivity exhibited by RNase A (Burgers and Eckstein,

1979).

Enzyme ko /k R p k o /k g p Rp/Sp

WT 42 3.1 10^ 7600

R69C-AA 40 1.6-10" 4000

R69C-TAA 38 1.2-10^ 3000

R69C-PA 35 3.9-10- 11

R69C-EA 36 1.1-10^ 3.0

R69K 34 2.7-10' .80

Table 4.2. Non-bridging thio-effects and stereoselectivity of PI-PLC with different side- chains at position 69.

Overall, presented data clearly demonstrate two important points: (i) positively charged side chains in the position 69 interact exclusively with pro-S oxygen of the phosphate group; (ii) there is a significant advantage of a bidentate fimctional group at position 69 for the catalysis by bacterial PI-PLC.

Combination of the data presented here with the available structural (Heinz et al.,

1995) and fimctional information (Gassier et al., 1997; Hondal et al., 1997b; Hondal et 85 al., 1998; FCubiak et al., 1999) on the bacterial PI-PLC suggests several possible arrangements for the interaction of Arg69 with the substrate during the catalysis (Figure

4.5), which would help to explain the necessity for the bidentate functional group.

First (Figure 4.5, A), in the most straightforward design, both terminal nitrogens of the arginine are hydrogen-bonded to the pro-S oxygen of the phosphate. It is not unusual for the non-bridging oxygens of phosphate groups to receive more than one hydrogen bond, and such an interaction is expected to be favorable with positively charged species, such as guanidine group (Hannon and Anslyn, 1993; Salunke and

Vijayan, 1981)

However, it is not clear how this second hydrogen bond will contribute to the catalysis, particularly if one takes into account that the transphosphorylation appears to proceed through slightly dissociative transition state (Chapter 5) with very little, if any, of additional negative charge developing on the non-bridging oxygens. Besides, presence of the second hydrogen bond contributes at least 10-100 fold more to the rate acceleration than presence of the first one (Table 4.2). This points out to the cooperative relationship between the two hydrogen bonds as opposed to the additive one as would be expected from the arrangement under consideration. In conclusion, while the interaction of both nitrogen atoms with pro-S oxygen can not be ruled out completely, it fails to give adequate explanations to the available data.

86 His32 Arg69

Asp33 O O H—I " i i His82

His32 Arg69

Asp33 O O H—I B

Asp274 His32 Arg69

o O H—I ill

. i (j- H -

Figure 4.5: Possible arrangements for the Arg69’s bidentate interaction with the substrate and other components of the active site during catalysis: (A) both p-nitrogens are hydrogen-bonded to the pro-S oxygen of the phosphate; (B) interaction with pro-S oxygen and the general acid diad, Asp33—His82; (C) interaction with pro-S oxygen and 2-OH of inositol, nucleophile in the reaction. 87 Second possibility (Figure 4.5, B) is based on the established interaction between

arginine-69 and general acid diad, Asp33—His82 (Kubiak et al., 1999). It should be

emphasized that the arrangement on Figure 4.5, B implies that second r;-nitrogen atom of

the arginine-69 is needed exclusively for the interaction with the diad and its absence

perturbs the diad’s function. Total contribution from the general acid catalysis to the

catalytic power of the bacterial PI-PLC is estimated at 10^-10^ (Gassier et al., 1997;

Hondal et al., 1998), so even relatively minor perturbation of the diad’s function would

have a profound effect on the catalysis.

In order to corroborate this possibility, we used bridging thio-effects to check if

general acid’s function is intact in the modified proteins. As it was shown earlier (Hondal

et al., 1997a), mutants of PI-PLC with disrupted general acid function had a “reversal” of

bridging thio-effect, i.e. ko/ks < 1. This phenomenon is due to the fact that thiolate has

lover pKa and is a better leaving group, which requires no assistance from a general acid.

Comparing activities of WT, R69C-AA, and R69C-PA toward DPPsI (Table 4.2) and

DOsPI (data not shown) gave bridging thio-effects of 25, 50, and 50, respectively.

“Normal” thio-effects for both arginine and lysine analogs indicate that in either case

function of the general acid has not been perturbed. Therefore, presence of the second q-

nitrogen of arginine-69 does not contribute to the stabilization and/or orientation of the general acid diad, as suggested by Figure 4.5, B.

In the crystal structure of PI-PLC complex with wyo-inositol Arg-69 was shown to be hydrogen-bonded to 2-OH of inositol (Heinz et al., 1995) (Figure 1.6), thus it is

88 possible that during catalysis arginine-69 interacts with both pro-S oxygen of the phosphate and 2-OH of inositol (Figure 4.5, C).

Mechanistic grounds for the double interaction. Proposed interaction of arginine-

69 with 2-OH of inositol would lower pfQ of the incoming nucleophile (Vishveshwara et al., 1998), facilitating proton abstraction by His32, and would help to bring the hydroxyl and phosphate into proper orientation for the catalysis. It was shown in model studies with different classes of intramolecular reactions (Jencks, 1975; Page and Jencks, 1971 ) that fixing reacting groups in the orientation most favorable for the reaction (termed by T.

Bruice and corworkers “near attack conformation” or “NAC” (Bruice and Lightstone,

1999)) could result in rate increase up to 10^ fold. Furthermore, this proposed interaction will explain the unusually high thio-effects observed for Sp-DPPsI since in such an arrangement introduction of sulfur atom would disrupt not only the interaction with the phosphate, but also the orientational effect of the arginine. Consequently, these results add one more factor to consider when one attempts to interpret large thio-effects in an enzymatic reaction (Herschlag, 1994).

Even though some alternatives can not be ruled out completely, interaction with both phosphate and 2-OH of inositol seems to be the most likely arrangement for the

“bidentate” function of Arg69, having support from structural, functional, and model data. Although it is not unusual for an arginine in the active site of an enzyme to interact with two functional groups of its substrate (see, for example (Calnan et al., 1991; Hannon and Anslyn, 1993; Sondek et al., 1994)), proposed interaction with both phosphate and the incoming nucleophile seems to be a unique feature of the bacterial PI-PLC.

89 R69A and R69G mutants of the PI-PLC are activated by guanidine chloride

(Figure 4.6), but the achieved activity is even lower than that of R69C-PA modified

enzyme. Activation by guanidine is highly specific for those mutants: neither simple

amines nor guanidine analogs activate them to any appreciable extent (data not shown).

These results emphasize the fact that guanidine group by itself is not enough for the

effective catalysis, it needs to be covalently anchored in the active site. Such positioning

of the catalytic group in an enzyme’s active site could accelerate catalysis by at least one

thousand fold (Admiraal et al., 1999).

Comparison with RNase A.Numerous similarities has been found between

catalytic mechanisms of RNase A and bacterial PI-PLC. Whereas substrates for both

enzymes are very similar, inositol phosphodiesters are at least 10^ times more stable in

imidazole buffers than corresponding ribose phophodiester (Kubiak, 1999). Even though

reason(s) for this higher chemical stability is not immediately apparent, it might explain

why PI-PLC must employ much more elaborate mechanism to achieve similar catalytic

power. In RNase A lysine-41 was assigned the fimction of phosphate activation

(Blackburn and Moore, 1982; Richards and Wyckoff, 1971). In the work similar to the one presented here, RNase A was shown to prefer lysine and its analogs at the position 41

(Messmore et al., 1995). However, the difference between “lysine” and “arginine” series was not as dramatic as in the present work and could be attributed, at least in part, to a steric effect resulting fi’om replacement of amino group by much bulkier guanidine or amidine group.

90 08

O ) 0.6

>. > O 0.4

0.0 0 20 40 60 80 100120 140 160 180 [Gnd], mM

Figure 4.6: Activation of R69A (• ) and R69G (▲) mutants by guanidine chloride. Solid lines are results of non-linear fit of each set of data to the Michaelis-Menten equation with the following parameters: R69A - Vma.x = (0.81±0.03) U/mg, K

Comparison with mammalian PI-PLC. Crystal structure of mammalian PI-PLC 51 was solved in complex with 1,4,5-mio-inositol triphosphate (Essen et al., 1996), which gives somewhat clearer picture of 1-phosphate’s interactions in the active site (Figure

1.5). There are two major differences from the active site of the bacterial enzyme: arginine is replaced by a carboxylate (Asp343) with calcium ion and His311

(corresponding to bacterial s His32) is moved from 2-OH toward pro-S oxygen of the phosphate. Calcium is coordinated by 2-OH of inositol and pro-S oxygen of the phosphate. Comparison with the proposed model for the bacterial enzyme (Figure 4.5, C) reveals that in mammalian PI-PLC arginine’s function is replaced by combined effort of the calcium ion and His311, where calcium takes over bridging between 2-OH and

91 phosphate (and, most likely, lowers the pKa of the former) while His311 contributes to the stabilization of the negative charge on the phosphate.

In summary, site-directed chemical modification along with the use of the phosphorothioate substrate analogs allowed us to propose a dual function for the catalytic arginine and to get further insight into the mechanism of bacterial PI-PLC with an emphasis on how the enzyme compensates for the intrinsic chemical inertness of its substrate. Present work has also demonstrated that a significant portion of non-bridging thio-effects in an enzymatic phosphoryl transfer reaction could be attributed to the factors other than a direct interaction between a phosphate group and an enzyme, which has important implications for the interpretation of the thio-effects in enzymatic reactions.

92 CHAPTER 5

PHYSICO-CHEMICAL CHARACTERIZATION OF INTRAMOLECULAR

PHOSPHORYL TRANSFER IN INOSITOLPHOSPHODIESTERS

5.1 INTRODUCTION

5.1.1 Reactions o f phosphate esters in chemistry and biology

Reaction of phosphoryl transfer is most likely the most ubiquitous one in

biological systems (Westheimer, 1987): energy storage and release, signaling,

metabolism, manipulations of the genetic material, all these vital processes involve

reactions of phosphate esters. As pointed out by Thatcher and Kluger (Thatcher and

Kluger, 1989), “phosphoryl transfer” and “phosphate transfer” are not the best names for

the reaction in question, but for historical reasons these terms have become widely

accepted in literature and are being used in this work along with several synonyms.

Another important aspect of the phosphate chemistry arises from the fact that

many active ingredients in chemical weapons, pesticides, and herbicides are phosphate

esters. Fast and effective detoxification of those compounds by chemical and biological

means has become a high priority recently due to the intensified use of chemicals in agriculture and the reduction of chemical weapons’ stockpiles worldwide.

93 o

NuO...... P...... OLg

O

NuO V P OLg O

NuO "V ""P OLg

Figure 5.1 : Classification of the transition states in the phopsphoryl transfer reactions, based on changes in the bond order to phosphorus atom relative to the ground state: dissociative (decrease in the overall bond order); Sx2-like (no change in the overall bond order); associative (increase in the overall bond order).

94 Reaction catalyzed by PI-PLC belongs to the very important class of phosphoryl transfer reactions: intramolecular reaction with the formation of cyclic product or intermediate. Knowledge from studies of the chemical and enzymatic mechanisms of this reaction will provide insights into reactions of ribonucleases (Raines, I998)(see also

Chapter 1), ribozymes (Cech et al., 1992), and adenylate cyclases (Jaworska-Adamu and

Cybulska, 1991).

5.1.2 Linear free energy relationships: probes of the transition states for chemical and enzymatic reactions

Pentacoordinate transition states (T.S.) of phosphoryl transfer are classified on the basis of the degree of bond formation to the incoming nucleophile and the degree of bond breaking to the leaving group (Figure 5.1). Available data indicate that there is a rough correlation between type of the phosphate ester and the T.S. of the reaction: monoesters - dissociative, diesters - Sn2, triesters - associative (Herschlag and Jencks, 1989).

In order to obtain information about transition states in enzymatic and imidazole- catalyzed reactions of inositol phosphodiestes we used linear free energy relationships

(LEER), which is empirical linear relationships between activation free energies (AG*) and equilibrium free energies (AG°) for the reactants and products or some intermediate

(Fuchs and Lewis, 1974; Johnson, 1975; Kresge, 1974; Shorter, 1969; Wold and

Sjostrom, 1978). Probably the most common LEER is Bronsted relationship (sometimes called “Bronsted law”) which correlates rate of a reaction with the pK, of a reaction component, as a rule - a leaving group or a nucleophile:

log[rate] = PxpKa + C,

95 where p is Brensted coefficient and C is an integration constant. P provides measure for

the degree of bond formation or breaking in the transition state.

LFER is a valuable tool in studying transition states o f chemical reactions, but its

application to enzymatic reactions have significant uncertainties and limitations

(Hollfelder and Herschlag, 1995; Kirsch, 1972), some of which will be illustrated by the results presented here.

5.2 MATERIALS AND METHODS

D-wyo-inositol was from Sigma. p-Cresol, phenol,, 4-chlorophenol, 2- fluorophenol, 3,5-dichlorophenol, and p-nitrophenol were from Aldrich. Organic solvents were from Fisher and Aldrich. All other reagents were of the highest grade available commercially.

2,3,4,5,6-penta(methoxymethylene)-lD-wyo-inositol (Scheme 5.1,1) was synthesized as described previously (Bruzik and Tsai, 1992).

5.2.1 Synthesis o f aryl phosphoinositides (3).

The synthetic procedure is shown in Scheme 5.1.Typically, to the mixture of

2,3,4,5,6-penta(methoxymethylene)-lD-/Myo-inositol (1, 400 mg, 1.0 mmol) and diisopropylethylamine (450 pL, 2.6 mmol) in dry chloroform (1 mL) was added O- methyl phosphorodichloridite (110 pL, 1.1 mmol) at -50 °C with stirring. After 8 h, a solution of a dry phenol (1.1 mmol) was added to this mixture, and stirring was continued at

96 1

OR OR OH RO, OH RO, ,0—P—OMe HO, Mil iv-v o(S) RO OR RO OR HO OH OR OR OH

Scheme 5.1 : Synthesis of aryl inositides. R, methoxymethylene; i, ChP-OMe, iPriEtN; ii, phenol or a substituted phenol (for the complete list see text and Figure 5.2), iPrzEtN; iii,

(But)4NI0 4 for Y =0 and Sg for Y=S; iv, MesN; v, EtSH/BFs-EtiO. room temperature for 4 h. After this step, reaction mixture was split into two portions, one for oxidation, and another for sulfurization. Oxidation was done by addition of tetrabutylammonium periodate at -20°C with subsequent stirring at room temperature for

2 hrs. For the sulfurization, after solvent removal and addition of sulfur, dry CS? (2 mL) was added and the resulting suspension was stirred at room temperature for another 10 h.

The following steps were the same for both phosphate and phosphorothioate compounds.

Evaporation of the solvent followed by chromatography on silica gel using hexane- acetone (6:1, v/v) as the eluent gave the triester 2 (typical yield 60-70%) as a colorless oil. The tri ester 2 was dissolved in dry liquid trimethylamine (1 mL) at -10°C, and the resulting solution was stored at room temperature for 16 h. The amine was evaporated, and the residue was dissolved in dry ethanethiol ( 1 mL) and added with boron tri fluoride etherate. After 40 min all solvents were evaporated, and the residue was

97 chromatographed on silica gel using chloroform-methanol-acetic acid (6:4:0.1, v/v) as the

eluent to yield pure aryl inositide (3) as colorless or pale yellow solid.

Structure and purity of all compounds were verified by 'H and ^’P-NMR.

5.2.2 ^‘P-NMR assays:

All ' P-NMR assays were performed on DRX-500 and DRX-600 NMR spectrometers (Bruker) equipped with broad band probes and temperature-contro 1 units.

All spectra were acquired with inverse-gated proton decoupling to avoid a build up of heteronuclear NOEs. In order to prevent signal saturation, pulses of no more than 30° and relaxation delays of at least 2 seconds were used. All samples contained inorganic phosphate of known concentration (typically - 5 mM) as a standard for quantitative determination of the reaction components. Specific conditions and data analyses were as follows:

aj enzymatic reactions

Reactions were done in 0.5 ml of 50 mM MOPS buffer, pH 7.5, at 25°C.

Substrate concentration was 20-25 mM, which is believed to be saturating

concentration for this type of the substrate (Rukavishnikov et al., 1997).

Reactions were initiated by addition of 5-20 pi solution of the WT enzyme of the

appropriate concentrations. Initial rates were obtained fi-om the linear portions of

IcP vs. time plots and expressed in pm olxmin'x(mg of protein)'*;

b) nan-enzymatic reactions

Reactions were done in 0.6 ml of 0.5 M imidazole, pH 7.0, at 80°C. Initial

concentration of substrate was 6-8 mM. Reactions were initiated by bringing

98 sample temperature to 80°C (although reaction rate at RT was negligible, time

between dissolving substrate and reaction initiation was kept to a necessary

minimum, especially for the substrates with low pKaS of the leaving groups).

Reactions’ progress was followed for at least two half-lives. Pseudo first order

rate constants were obtained from non-linear fits to substrate vs. time plots to the

integrated form of the first order rate law using SigmaPlot (Jandel Corp.).

5.2.3 Purification o f Rp phosphorothioates

Reactions with Sp components of phosphorothioate substrates were driven to completion by sample dilution and/or by addition of fresh portion(s) of the WT enzyme as monitored by P-NMR. Subsequently, samples were lyophilized, resuspended in chloroform-methanol-acetic acid (2:1:0.03, v/v) and applied on silica gel column (20 ml bed volume). Rp aryl thiophospoinositols were eluted with chloroform-methanol-acetic acid (1:1:0.02, v/v). Organic solvents were removed by evaporation and samples were freeze-dried twice from water to remove the residual acetic acid. Recovery of Rp phosphorothioates after purification was approximately 70%.

99 pK,=13.7 HOHO Imidazole A" —roH HO ..... V OH K O Ç « * \ _ 0 H NPIP "o -X^— OH

HO O , Ü Imidazole NPUP 'A' ■ p ;— 9 2H OzZf-/ -O-P- pK .»1i3

Figure 5.2: Comparison of imidazole-catalyzed reaction of ribosyl and inositol phosphodiesters. Inositol data are from (Kubiak, 1999) and ribosyl data are from (Davis et al., 1988a).

5.3 RESULTS AND DISCUSSION

We have known for a while that phosphatidyl inositols are much more stable in imidazole buffers than corresponding ribosyl phosphodiesters (Figure 5.2). Two substrates have somewhat different pKaS of the respective nucleophilic OH group, but this difference is not big enough to explain almost one thousand-fold difference in the reaction rates. Different transition state for these two reactions is one of the possible explanations for the observed difference in reactivity.

Tri ester-like mechanism proposed for the RNase A (Anslyn and Breslow, 1989;

B res low and Chapman, 1996) was disputed on the grounds of small thio-effects for this enzyme (Herschlag, 1994). Protonation of non-bridging phosphate oxygen by a histidine

100 is very unlikely in the bacterial PI-PLC since its histidines seem to be anchored by their neighboring aspartates (Heinz et al., 1995; Hondal et al., 1998). On the other hand, available data do not exclude a possibility of complete proton transfer to pro-S oxygen atom from the Asp69 side chain in the transition state (Kubiak et al., 1999), rendering

T.S. of the enzymatic reaction more associative.

Structures of the transition states in both non-enzymatic and enzymatic reactions were probed by LFERs with inositol phosphodiesters carrying leaving groups with various pK^s.

Aryl inositol phosphodiesters were synthesized with pKaS of the leaving groups ranging from 7.1 to 10.2 (Figure 5.3). Same series o f compounds were made with phosphorothioates, where sulfur replaces one of non-bridging oxygen atoms, giving Rp and Sp isomers. It should be noted that due to the priority change in aryl inositol phosphorothioates, the absolute configurations of Rp and Sp isomers in these compounds are opposite of those in inositol phosphorothioates with DAG as a leaving group.

Therefore, in the aryl compounds Sp-phosphorothioate is a preferred substrate for bacterial PI-PLC.

101 CH, A

10.2 9.95 OH OH

OH

8.18 OH

Figure 5.3: Structures of aryl inositol phosphates and thiophosphates, used in the LFER studies: general structure and aryl leaving groups with their respective pKaS (pKa values are taken from (Davis et al., 1988a; Hollfelder and Herschlag, 1995)).

102 5.3.1 Non-enzymatic reactions o f inositol phosphodiesters

Non-enzymatic reactions were studied in 0.5 M imidazole (pH = 7.0) at 80°C to ensure reasonable rates for slower reactions. Imidazole as a catalyst is thought to be a good model for the enzymatic reaction, where histidine side chains are crucial for the catalysis. Typical time courses for the reactions as monitored by ^’P-NMR are shown on

Figure 5.4 for one each of phosphate and thiophosphate compounds. Reactions proceed exclusively through intramolecular cyclization, with 1,2-IcP (IcPs) being the major product and 1,6-IcP (IcPs) being the minor one, compounding to 10-15% of the total.

There were no significant desulfurization or aryl phosphate migration observed for all compounds. Significant hydrolysis of IcP was observed only in the reactions with p- cresol and phenol phosphodiesters. In a separate experiment, pseudo first order rate constant for the IcP hydrolysis was found to be 1.01x10"* min'* imder the same conditions, which is very similar to the rate constants of the two above compounds. No hydrolysis of cyclic thiophosphates was observed under the experimental conditions.

Pseudo first order rate constants of the intramolecular transphosphorylation were obtained from non-linear fit to the decay of the starting material (Figure 5.5) and are summarized in Table 5.1.

Reaction rates of the compoimds with the same leaving group are virtually identical for either phosphate or both phosphorothioate isomers. The only exception seems to be the Sp-thiophosphate with the p-nitrophenol as the leaving group. The rate constant for this compound is about 25-30% smaller than those of its peers. Although the reason for the difference is not clear, it is definitely not significant enough to bring any uncertainties to the interpretation of the results.

103 B

2 1 4 3 ...... 11. 6160 min I 182.7 min

4755 min 144.5 min

3490 min JiL 101 min

2130 min 57.5 min

770 min 13.9 min

ppn 3i p ppa 70 3lp

Figure 5.4: ^'P-NMR spectra of the representative time courses of the imidazole- catalyzed transestérification of aryl inositides: (A) reaction of phenol inositol phosphate; signals: * - inorganic phosphate (internal standard), 1 - starting material, 2 - 1,2-IcP, 3 - 1,6-IcP, 4 - 1-IP and 2-IP; (B) reaction of 3,5-dichlorophenol inositol phosphorothioate; signals: 1 - Sp isomer, 2 - Rp isomer, 3 - 1,2-c/j-IcPs (product of Rp isomer), 4-1,2- 7ra/75-IcPs (product o f Sp isomer), 5 -1 ,6 - cis- and /ra/ij-IcPs. Reaction conditions are given in Materials and Methods. 104 8

7

6

CL 4 O c (U 3 Q.

2

1

0 0 2000 6000 80004000 10000 Time, min

8

7

6

5

4

3

2

1

0 0 50 100 150 200 Time, min

Figure 5.5: Time courses of the imidazole-catalyzed transestérification reactions of (A) phenol inositol phosphate and (B) 3,5-dichlorophenol inositol phosphorothioate (Rp isomer). Solid lines are results of the non-linear fit to the integrated form of the first order rate law.

105 Pseudo first order rate constant, k'xlO^, min ’ Leaving group pKa Phosphate Sp- Rp- thiophosphate thiophosphate p-nitrophenol 7.14 170 ±6 122 ±2 152 ±6

3,5-dichloro­ 8.18 72 ±2 61 ±3 69 ±3 phenol 2-fluorophenol 8.80 ND 8.0 ±0.2 8.1 ±0.2 p-chlorophenol 9.38 4.6 ±0.1 4.62 ±0.09 4.45 ±0.07

Phenol 9.95 1.85 ±0.03 1.91 ±0.03 1.80 ±0.04 p-cresol 10.20 1.35 ±0.02 1.35 ±0.03 1.25 ±0.02 ND - not determined

Table 5.1. Rate constants of imidazole-catalyzed intramolecular transestérification of aryl inositol phosphodiesters

These results are in a sharp contrast to the similar studies on uridine 3'-(aryl phosphorothioate)s (analogs of RNA) (Oivanen et al., 1995), where thio-efleets were found to be around 2 for both isomers of phosphorothioates.

Given almost identical rate constants in all three series of compounds, it is not surprising that phosphate and both thiophosphate isomers give indistinguishable Brensted plots (Figure 5.6). As a result, phosphate and both isomers of thiophosphate compounds do not show any significant differences in Bronsted coefficients. Pig o f-0.7 indicates slightly dissociative transition state. This number is very similar to the previously

106 en

7 8 9 10 pKg of the leaving group

Figure 5.6: Bronsted plots of the imidazole-catalyzed transestérification of aryl inositol phosphates and phosphorothioates: phosphates (•), Sp-phosphorothioates (A), and Rp- phosphorothioates (■).

4

3

2

1

0 O) o -1

-2

■3

-4 7 8 9 10 pKg of the leaving group

Figure 5.7: Bronsted plots of the enzymatic transphosphorylation of aryl inositol phosphates and phosphorothioates catalyzed by WT PI-PLC: phosphates (•), Sp- phosphorothioates (A), and Rp-phosphorothioates (■).

107 reported value o f-0.54 for ribosyl phosphates (Davis et al., 1988a) and -0.64 for ribosyl

phosphorothioates (Aimer and Stroemberg, 1996).

The above results indicate that imidazole-catalyzed reaction of inositol phosphate

diesters proceeds through a transition state very similar to that of ribosyl phosphodiesters.

More importantly, this study demonstrates that introduction of the sulfur atom at the non­

bridging position of phosphate do not change intrinsic chemical reactivity of inositol

phosphate diesters. Therefore, all thio-effects observed in the enzymatic reactions (see

Chapter 4 and results below) are determined exclusively by substrate-enzyme interactions

and/or disruption of thereof.

As to the higher inertness of the inositol phosphodiester compared to ribosyl

phosphodiesters (which has been confirmed by the results presented above): most likely it

is a consequence of several factors, such as pKa of the nucleophile, small change in the

structure of the TS, and probably some others, each bringing a relatively small, but

additive, contributions.

5.3.3 LFER investigation of the enzymatic reaction.

Accumulated experience in the application of the LFER analysis to enzymatic

phosphoryl transfer reactions (Herschlag and Jencks, 1989; Thatcher and Kluger, 1989)

allows to formulate several conditions, which need to be met in order for such analysis to

be successful (Hollfelder and Herschlag, 1995). First, chemical step of the reaction

should be rate-limiting, otherwise leaving group (and/or nucleophile) dependence is not

going to be expressed in the observed rate. Second, studied enzyme should have as little as possible interaction with the group being altered in the LFER investigation. If this is

108 not the case, the rates of the enzymatic reactions could be affected by the structural changes in the leaving group or nucleophile rather than by their pKaS. Third, since second condition is almost never completely realized, compounds used in the enzymatic studies should be structurally homogeneous and their number should be large enough to provide an averaging off of any advertent effects.

For bacterial PI-PLC, no information is available on the rate-limiting step (Bruzik and Tsai, 1994). Fortunately, sulfur substitutions on the phosphate have a profound effect on the rate of the enzymatic reaction (Hondal, 1997; Hondal et al., 1997b), therefore phosphorothioates can be used to ensure that chemical step is rate-limiting.

Interaction with the leaving group for this enzyme is not specific (Bruzik et al.,

1992), but it is very important for the catalysis (Kubiak, 1999; Kubiak et al., 1999). This was taken into account in the design of substrate analogs, which did not have bulky groups in positions 2 and 6 of the benzene ring.

Table 5.2 shows activities of the WT PI-PLC toward the same sets of compounds that have been used in the imidazole-catalyzed reactions. As expected, enzymatic activity is significantly different between phosphate and two isomers of phosphorothioate compounds. Overall, thio-effects are very similar to those observed with DPPsI (Chapter

4), particularly for substrate analogs with higher pKa of the leaving group.

Interestingly, enzymatic reaction rate of p-nitrophenol Sp-phosphorothioate is (as also the case for the non-enzymatic reaction) lower than it would be expected judging from the trends among other analogs.

109 Vmax of WT PI-PLC, Leaving group pKa umolxmin*‘x(mg of protein)’

Phosphate Sp- Rp- thiophosphate® thiophosphate p-nitrophenol 7.14 1450 290 1.4x10’

3,5-dichloro­ 8.18 962 345 6.3x10- phenol 2-fluorophenol 8.80 245 26 1.1x10- p-chlorophenol 9.38 199 13.5 3.7x10"

Phenol 9.95 67.9 2.1 6.1x10"* p-cresol 10.20 82.5 4.5 8.8x10"* Absolute configuration of Sp aryl inositol phosphorothioate is the same as that of Rp-

DPPsI.

Table 5.2. Maximal activities of WT PI-PLC in the intramolecular transphosphorylation of aryl inositol phosphodiesters. Given are average values from at least two independent experiments.

Another anomaly among the analogs is revealed when activities with p-cresol and phenol leaving groups are compared. Despite its higher pKa (by 0.25 pKa units), p-cresol esters are better substrates in all three series of compounds. This behavior could be explained by the higher hydrophobicity of the p-cresol, allowing for better “hydrophobic” activation than other leaving groups. This activation appears to be enough to compensate for the small difference in the pKa with the compounds carrying phenol leaving group.

110 Bronsted plots of the enzymatic reactions (Figure 5.7), unlike imidazole-catalyzed ones, are very distinct for each of the three series of compounds. Phosphate esters have the smallest slope and the least scattered points in their plot. Both isomers of phosphorothioates have similar slopes and significantly more scattering in their data points. Bronsted coefficients o f the enzymatic and, for comparison, non-enzymatic reactions are compiled in Table 5.3.

Bronsted coefficient for the leaving group, Pig

Non-enzymatic reaction Enzymatic reaction

Phosphate -(0.74±0.07) -(0.46±0.06)

Sp-phosphorothioate -(0.68±0.07) -(0.72±0.15)

Rp-phosphorothioate -(0.72±0.07) -(0.82±0.09)

Table 5.3. Summary of Bronsted coefficients for non-enzymatic (imidazole-catalyzed) and enzymatic (WT PI-PLC) intramolecular transphosphorylation of aryl inositol phosphodiester.

As already mentioned above, all three coefficients in the non-enzymatic reaction are essentially the same. In the enzymatic reaction, Bronsted coefficients of phosphorothioates are very similar to each other and to those in the chemical reaction. On

111 the other hand, (3ig of phosphate analogs in the enzymatic reaction is significantly less negative than any other coefficient, enzymatic or non-enzymatic.

Less negative (3,g value in the enzymatic reaction with phosphate analogs can not serve as an evidence of more associative character of the T.S. since effect of pKa could be masked by protonation of the leaving group and/or by the fact that chemical step is not rate-limiting (Hollfelder and Herschlag, 1995). Since both isomers of thiophosphate return to the “normal” value of -0.7 for the Bronsted coefficient in the enzymatic reaction, most likely chemical step is not rate-limiting in the reactions with the phosphate esters. Use of slower substrates (including thiophosphates) is a common technique in

LFER studies of enzymatic reactions (Cole et al., 1994). Therefore, presented data argue for the transition state of the enzymatic reaction being very similar to that of the non- enzymatic reaction.

Lower slope in LFER of the RNase A reaction with aryl phosphates was attributed to the electrophilic catalysis by the enzyme (Davis et al., 1988b). This interpretation could have been applicable to the PI-PLC if both phosphate and Sp- thiophosphate series had less negative Bronsted coefficients. Slope would have been expected to return to non-enzymatic reaction’s value only for the Rp-thiophosphates since in this case interaction of the Arg69 and phosphate group is dismpted.

General acid catalysis has been shown to play very important role in the mechanism of bacterial PI-PLC (Gassier et al., 1997; Hondal et al., 1997a; Hondal et al.,

1998), thus it is surprising that it is not manifested in the enzymatic LFERs through lowering slopes of the Bronsted plots. At least two explanations are possible: first, substrates with aryl leaving group and/or with non-bridging sulfur could interfere with

112 the general acid function of the enzyme. This notion is in accord with the hypothesis

about the possible role of the hydrophobic leaving group in the rearrangement of the catalytic residues (Chapter 2, Figure 2.1). Second, protonation of the leaving group might occur after the rate-limiting step of the reaction. In all mechanisms proposed and discussed for the PI-PLC (Bruzik and Tsai, 1994; Gassier et al., 1997; Heinz et al., 1998;

Heinz et al., 1995; Hondal et al., 1998) it has been assumed that the T.S. is highly concerted, i.e. nucleophile deprotonation, nucleophilic attack itself, departure of the leaving group, and its protonation all occur at the same time. However, there is no evidence so far against multi-step chemistry in the enzyme’s active site.

In summary, LFER analysis was applied to non-enzymatic and PI-PLC catalyzed intramolecular transphosphorylation of inositol phosphate diesters. Transition states of both reactions were found to be essentially the same. Use of phosphothioate analogs allowed much more detailed interpretation of data than phosphate compounds alone.

Chemical reactivity of inositol phosphorothioates was found to be identical to inositol phosphates, thus establishing that all thio-effects observed in PI-PLC reactions are of enzyme-specific origin.

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