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Pyruvate Carboxylase

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Pyruvate Carboxylase PYRUVATE CARBOXYLASE: STUDIES ON THE REACTION MECHANISIU A THESIS SUBMTTTED BY NELSON FRÄNCTS BONIFACE PHTLLIPS, M.SC. To: THE UNTVERSTTY OF ADELAIDE SOUTH AUSTRALIA FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Department of Biochemistry University of Ade1aide, South Australia. December, 1981. \-- CONTENTS Page SUMMARY l_ STATEMENT v ACKNOWLEDGEMENTS vi ABBREVIATTONS vii CHAPTER I INTRODUCTTON I A. General properties of Biotin-containing Enzymes t I.A. I Reaction mechanism I I B. Chemistry of pyruvate Carboxylation 3 I.8.1 Carboxylation of Biotin 3 I.8.2 Specificity for Bicarbonate 5 I.8.3 Effect of Monovalent Cations 6 I.8.4 Activation by Acetyl CoA 7 I.B.4a Enzyme inactivation upon dilution and protection by acetyl CoA I I.B.4b Effect of CoA acetyl on catalytic activity t0 ï.C. Pyruvate Carboxylase Reaction Sequence 13 I.C.1 The Non-classical ping-pong Reaction pathway I5 f.C.2 _The Sequential Mechanism L7 I.D. Mechanism of the First partial Reaction 20 I.D.la The concerted mechanism 2T I.D.Ib The enzyme activation mechanism 22 I.D.lc The substrate activation model 24 f.E Mechanism of Transcarboxylation 26 r.F. variable stoichiometry of pyruvate carboxylation 28 I.c. Chemistry of the Active Site 29 I.c.1 Chemical Modification 30 I.G.2 Protein Sequence Determination 32 I.H . lHE AIMS OF THE PROJECT 34 CHAPTER II MATERTAIS AND METHODS IT.A MATERIALS 36 II.A.1 General Chemicals 36 lI.A.2 Radioactive Chemicals 37 II.A.3 Enzymes and Proteins 37 IT.B. MSTHODS 38 II.B.1 Preparation and Purification of Nucleotides 38 II.B.Ia Acety1 CoA 38 rr.B.rb ¡y-32e1are 38 II.B.2 Purification of Pyruvate Carboxylase 39 II.B.3 Storage of Pyruvate Carboxylase 40 II.B.4 Protein Estimation 40 II.B.5 Determination of Radioactivity 40 II.B.6 Measurement of Pyruvate Carboxylase Activity 4L II.B.6a The Coupled spectrophotometric assay system 4I II.B.6b The radiochemical assay system 42 II.B.7 Separation of Radioactive Nucleotides and rnorganic Phosphate 4l II.B.8 Isotopic Exchange Reaction Assays 43 II.B.8a ATP:Pi Isotopic exchange reaction assay 43 II.B.8b ATP:ADP isotopic exchange reaction assay 44 II.B.8c HCO;:oxaloacetateJ isotopic exchange reaction assay 45 II.B.8d Pyruvate:oxal-oacetate isotopic exchange reaction assay 45 II.B.8e Calculation of rates of isotopic exchange exchange 46 II.B.9 Polyacrylamide Gel Electrophoresis 47 II.B.t0 S-Carboxymethylation of Cysteinyl Residues 48 ILB.11 Peptide Hydrolysis and Sequencing Procedure 48 II.B.lIa Amino Acid analysis 48 II. B.lIb Anallztical peptide maps 49 II.B.llc N-terminal sequence determination 49 II.B.12 Determination of Biotin Concentration 49 CHAPTER IIf MECHANISM OF THE FIRST PARTIAL REACTION III.A ÏNTRODUCTION 51 ÏII.B MATERIALS AND METHODS 55 III.B.I General Methods 55 IIï.E}.2 Assay Methods 55 III.B.2a Overall reaction assay procedures 55 III.B.2b Isotopic exchange reaction assays 56 III.B.2c Measurement of the rate of ATP hydrolysis 56 III.B.2d Assay of the enzl.rnic reaction ín the reverse direction 57 IIT.B.3 Data Processing 57 TII.C. RESULTS 58 )L III . C.1 Mgo'-dependent ATP:ADP isotopic exchange reaction catlaysed by chicken liver pyruvate carboxylase 58 ITT .C .2 Effect of HCO; on isotopic exchange reactions involving ATP 61 III.C.3 Effect of acetyl CoA on isotopic exchange reactions involving ATP 62 III.C.4 Slow i-sotopic exchange reactions and substrate synergism 66 III.C.5 Variability of the Mg''-dependent)L ATP:ADP isotopic exchange reaction rate 70 III.D. DTSCUSSION 7L CHAPTER IV ISOLATION OF THE PUTATIVE ?i--teNz- ëo2" BIOTIN COMPLEX IV.A INTRODUCTION 74 IV.B MATERIALS AND METHODS 75 IV.B.1 General methods 75 IV.B.2 Assay Methods 75 't ¿, IV.B.3 [-'Clcarboxyl Group Transfer to Pyruvate 75 rv.B.4 Transfer of t32pil from t32pilENZ-biotin complex to ADP 75 TV.C RESULTS 76 IV.C. Ia Isolation of carboxy-enzyme complexes 76 fV.c.Ib Transfer of tl4clcarboxyl group to pyruvate 77 IV.C.2a Isolation of the phosphoryl-enzyme biotin complex 78 3 2Pi\u*r- rV.c.2b Transfer of t32Pil f rom ë.oV.' biotin complex to ADP 82 IV.D DISCUSSION 85 CHAPTER V CHARACTERIZATION OF THE ISOLATED Z.BIOTTN COMPLEX V.A INTRODUCTION 87 V.B. METHODS 90 V.8.1 General Methods 90 V.8.2 Assay Methods 90 V.8.3 Preparation and Isolation of Radioactively Labellea filrNZ-biotin comprexes 91 ëoz" 32îj'\ENZ- v.B.4 Determinatiõn of harf-life of co'' biotin complex 9I V.8.5 Determination of half-life of ,rFi¡-rNz- ëor' biotin complex 9I V.B.6 Preparation of diazomethane 92 V.8.7 Preparation of Tryptic Peptides 93 V.C. RESULTS 93 V.C.I The Possibility of o-phosphobiotin-ENZ 93 V.C.1. I Trapping of enzyme-bound intermediate (s) with diazomethane 94 V.C.L.2 Stability of biotin to acid hydrolysis 98 v.c.2 srabiliry of fi;ætrr-biorin complex 98 cor' V.C.3 Attempts to trap and demonstrate carboxy- phosphate in the reaction solution 100 V.D. DISCUSSION 101 CHAPTER VI MECHANTSM OF ACETYL CoA ACTTVATION VI.A INTRODUCTION r05 VT. B I"IATERIALS AND METHODS 105 VI.B.l Assay Methods 105 1Ã VI.B.2 t-'Clcarboxyl group transfer reactions 105 VI.C RESULTS 106 VI.C.I Effect of acetyl CoA on the formation of ENZ-carboxybiotin and the Èranslocation process 106 VI.C.2 The rabsoluter requirement for acetyl CoA 110 VI.D DISCUSSTON 113 CHAPTER VII AFFINITY LABELLTNG AND ISOLATION OF TI]E Mg-oATP-PEPTIDE VTI.A TNTRODUCTION 115 VIT.B MATERIALS AND I"IETHODS 116 VIf-B.l Assay Methods 116 VfI.B.2 Synthesis of oATp 1't 6 VrI.B.3 Synthesis of tysyl- tu-14cl oATp 117 Vff .B.4 Covalent modification of the enz)rme 117 VIT.C RESULTS 11ì8 VIf.C.1 frreversible inhibition by oATp 118 vrr-c-2 Protection of the enzyme from inactivation by oATp 118 VfI.C.3 Isolation óf the oATp-peptide 119 VfI. C. 3 .1 preparation 119 VII.C.3.2 ldentification of the modified amino acid 120 VfI.C. 3. 3 Fractionation of peptides 121 VII.C.4 Other methods of purification 12t VIf .C-4.1 The charcoal-adsorption method 121 VII.C-4.2 The ion-exchange method 1Z+ VTI.D CONCLUSION 125 CHAPTER VITI GENERAL DISCUSSION '126 VITT. I The mechanism of the first partial reaction 126 vrrt.2 The nature of the isolated li->u*z-biotin col comprex 1,28 vrrr.3 The requirement of activation by acetyl CoA 'l3o VIII.4 The M9-ATP binding site 1t1 REFÀRENæS 15t l_ SU¡{MARY In view of the uncertainties in the previous interpretation of experimental data associated with the mechanism of ATp hydrolysis coupled to co2 fixation as catalysed by pyruvate carboxyrases, the kinetic evidence has been re-examined. As a result, a step-wise reaction mechanism has been proposed for the first partial reaction, viz¡ ENZ-biotin + ATp r-- ATp-ENz-biotin (i) ATP-ENz-biotin + HCoä P,i>nxz-biotin + ADp (ii) J coz-1'-- --u"u Dì +ç9!vl-cra ENZ-biorin-co, + pi (iii) coz*Ï;----u*r-biorin contrary to previous reports, the t'tg2+-dependent ATp:ADp exchange reaction was shown to be an integrar part of the reaction mechanism and has been incorporated. into the reaction pathway for the first partiar reaction. The exchange reaction in question was previousry considered to be the result of an abortive or kineticarry insignificant pathway; however rigorous attempts to remove endogenous Ievels of bicarbonate have shown that the exchange reaction v/as infruenced by bicarbonate. sigrirarry acetyr coA has arso been shown to influence the ir,tg2+-dependent isotopic exchange reaction between ATp and ADp. The probable involve- ment of an enzyme-bound carboxyphosphate (via eq. ii) is consistent with all of the avairabre kinetic data on the reaction mechanism. Further evidence that the overall pyruvate carboxylase catalysed reaction proceeds via a sequential l_l_. pathway, has been obtained by demonstrating a synergistic effect of the second partial reaction substrates on the fírst partial isotopic exchanqe reactions involving ATp. On the basis of the proposed mechanism, experiments were carried out to isolate the postulated n¡t-;"*Z-biotin comptex. By reacting Lhe enzyme with f r-t'rlÎôn in rhe absence of acetyl CoA, and subsequent gel filtration at :2e,i;>eNZ-biotin 4oc it was possible to isolate the ðõz ?-, complex. By its ability to transfer the [-'pi]phosphoryl group to ADP in a back reaction to form ATP, the complex was shown to be kinetically competent. Similarly reacting the enzyme with ntacor, enabled the isolation of a tlacl labelled complex tfaPltlENZ-biotin), by similar procedures. rne 14P,a-)xNZ-biotin complex was also shown to be kinetic- coz 1^ ally competent by its ability to transfer the t--cl carboxyl group to pyruvate in the presence of acetyl CoA. Although the carboxyl group on the complex was found to be more labÍIe than the phosphoryl group, the kinetic competency with respect to both the phosphoryl group and the carboxyl group of the complex r^/ere found to be similar, indicating that they \^rere part of the same complex. During attempts to characterize the isolated Pl1>eNz- io biotin complex, the possibility of an involvement of2a phosphorylated biotin derivative was considered.
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