5-Aminolevulinic-Acid Synthetase of Rhodopseudomonas sp heroides Y
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1 Eur. J. Biochem. 40, (1973) 5-Aminolevulinic-Acid Synthetase of Rhodopseudomonas sp heroides Y Kinetic Mechanism and nhibition by ATP Michitle FANCA-GAGNER and Jenny CLEMENT-METRAL Laboratoire de Photosynthese du Centre National de la Recherche Scientifique, Gif-sur-Yvette (Received May 17/Juy 27, 1973) The results of initial velocity studies on the 5-aminolevulinic acid synthetase reaction and product inhibition studies are consistent with an ordered bi-bi mechanism in which glycine binds to the enzyme first and 5-aminolevulinic acid dissociates last. The results are confirmed by the spectral changes of holoenzyme caused by the substrates. 5-Aminolevulinic acid synthetase activity is inhibited only by adenosine triphosphate, guanosine triphosphate and pyrophosphate. This inhibition is competitive with glycine and noncompetitive with succinyl-coa. Experiments with [W~~PATP, [y-32p]atp and [32P,]pyrophosphate have shown that adenosine triphosphate and pyrophosphate are bound on each of both forms of 5-aminolevulinic acid synthetase. The ratio is one mole per mole of enzyme. The estimation of -SH groups with 5,5 -dithiobis-(2-nitrobenzoic acid) after binding of inhibitor shows that one -SH is masked. After p-hydroxymercuribenzoate action on the enzyme, 5-aminolevulinic acid synthetase does not bind anymore [32P]ATP and is completely inactive. We suggest that ATP and pyrophosphate inhibit 5-aminolevulinic acid synthetase by acting on an -SH group of the active center of 5-aminolevulinic acid synthetase. 5-Aminolevulinic acid synthetase catalyses the reaction : G1ycine + succiny1 pyridoxal 5 -pllosphate CoASH + 5-Aminolevulinic acid + CO,. The kinetic mechanism has been partially described by Kikuchi et al. [] in a previous study with radioactive glycine and succinyl-coa. Akhtar and Jordan [2] have shown that succinyl-coa reacts with pyridoxal-glycine after liberation of H+ by the fixed glycine. Warnick and Burham [3] studied the mechanism with Cleland s method [4,5] only for the initial velocity studies; no study of mechanism of product inhibition and no sequence of events have been described. We have studied the kinetic mechanism, determined the kinetic apparent constants of the reaction and proposed a reactional sequence. Previously [6a], we have shown that only ATP inhibits the two fractions of Rhodopseudornonas spheroides Y 5-aminolevulinic acid synthetase about 750/,. ADP and AMP have no effect. n this report we confirm that only triphosphonucleotides and pyrophosphate are inhibitors. The kinetic mechanism of this inhibition and the mode of fixation of these inhibitors on the enzyme were investigated. MATERALS AND METHODS We used the holoenzyme of fraction prepared as previously described after the electrofocusing step l6bl. Kinetic studies of 5-aminolevulinic acid synthetase reaction were carried out by Urata and Granick s method [7] in which the initial velocity is measured by the formation of 5-aminolevulinic acid. nhibition by 5-aminolevulinic acid studies used Ellman s method [S] which follows the formation of CoA-SH (for details see [5]). The succinyl-coa was synthesized by the method of Simon and Shemin [9]. Concentration of succinyl- CoA was estimated by the method of Cha and Park [lo]. The estimation of -SH groups was carried out with Ellman s method [S] with purenative or denatured enzyme (denaturation by 0.5O/, sodium dodecyl sulfate, 4 M or 8 M urea). Molecular weight of the pure enzyme was taken as [6] for the two 5-aminolevulinic acid synthetases. To study the fixation of inhibitors, we used pure fraction and fraction 1 5-aminolevulinic acid synthetases homogenous in disc electrophoresis [6]. [32P]ATP or sodium [32P,]pyrophosphate fixation were studied by incubating [Y-~~P]ATP (1328mCil
2 20 5-Aminolevulinic Acid Synthetase from Rhodopseudomonas spheroides Y 6 0 [Glycine] (rnm- ) Fig. 1. nitial velocity pattern of 5-aminobvulinic acid synthetaae reaction. We have used the method of Urata and Granick [7] which assays the 5-aminolevulinic acid formation (1 U = 1 pmol x h-l). (A) Double-reciprocal plot with glycine as the varying substrate; succinyl-coa concentrations were: [Succinyl- CoA] (1M-l) (1) 200pM, (2) OOpM, (3) 65 pm, (4) 50pM. (B) Doublereciprocal plot with succinyl-coa as the varying substrate; glycine concentrations were: (1) 400 mm, (2) 200 mm, (3) 100 mm, (4) 50 mm, (5) 20 mm, (6) 10 mm nmol) or [cx-~~patp (966 mci/nmol) or sodium [32P,]pyrophosphate (32 mci/nmol) (50 nmol) with 5-aminolevulinic acid synthetase (0 nmol, mol. wt = ) in 0.1 M Tris-HC1 ph 8 for 30 min at room temperature. The reaction mixture was isolated by gel filtration on Sephadex G-200 using 0.1 M Tris ph 8 [ll. Fractions were assayed for protein by Lowry s procedure [12]. 32P was determined in 0 ml Bray s solution in a Packard scintillation Tri-carb counter [131. [y-32p]atp, [CX-~~PATP and sodium [32P2]pyrophosphate were purchased from CEA Saclay (France). RESULTS Analysis of Kinetic Mechanism. Kinetics of 5-Aminolevulinic-Acid-Xynthetase Reaction The effects of varied concentrations of glycine on the initial rate of the reaction are shown in Fig.1A represented in a double-reciprocal plot. The effects of increasing concentrations of succinyl-coa for different values of glycine are shown in Fig. B. The double-reciprocal plots converge at a common point on the left of the ordinate. These results indicate a sequential mechanism. Plots of the intercepts and slopes are linear functions of the appropriate reciprocal substrate concentrations, as is required by the low rate (cf. Cleland [4]). The kinetic constants are determined graphically and by Cleland s equations. They are given in Table 1 (we use Cleland s terminology) [4]. Table 1. Kinetic constants Substrates v K* Kia ~ pmol x h- x mg protein- mm Glycine * 4 54 * 1 Succinyl-CoA & The value of Ki, is the same whether glycine or succinyl-coa is the variable substrate and suggests that the mechanism is ordered bi-bi. Product-nhibition Studies Analysis of product-inhibition patterns of 5- aminolevulinic acid synthetase (Fig. 2) was undertaken to gain further information about the kinetic mechanism. Kinetic constants of inhibition of the reaction by CoA and by 5-aminolevulinic acid are given in Table 2. Cleland [4] has provided a convenient set of criteria to distinguish among various mechanisms for two-substrate enzymes, based on product-inhibition patterns. Of the various simple kinetic mechanisms considered by Cleland, only the ordered bi-bi mechanism is fully consistent in the case of 5-aminolevulinic acid synthetase with the product inhibition studies as inhibition by 5-aminolevulinic acid is competitive with glycine and the other product inhibition patterns are non-competitive.
3 ~~ M. Fanica-Gaignier and J. Clement-Metral 21 -._ c + aj 0 L 20 E" A J3 [Glycine] (rnm-') / [ Succinyl - CoA] Fig. 2. Product-inhibition patterns of 5-aminoleuulinic acid synthetase reaction. (1 U = 1 pmolxh-l). (A) nhibition by CoA, glycine is the variable substrate while succinyl-coa is held constant at 0.2 mm. The concentrations of CoA were: (1) none, (2) 0.2 mm, (3) 1 mm; we have used the method of Urata and Granick [7] for the assay. (B) nhibition by CoA, succinyl CoA is the variable substrate and glycine is held constant at 200mK The concentrations of CoA were: () none, (2) 0.2 mm, (3) 1 mm; we have used the method of Urata and Granick [7] for the assay. (C) nhibition by 5- aminolevulinic acid, succinyl CoA is the variable substrate and glycine is held constant at 200 mm. The concentrations of 5-aminolevulinic acid were: (1) none, (2) 0.2 mm, (3) 1 mm; we have used the method of Ellman [8] for the assay. (D) nhibition by 5-aminolevnlinic acid, glycine is the variable substrate and succinyl-coa is held constant at 0.2 mm. The concentrations of 5-aminolevulinic acid were : (1) none, (2) 0.2mM, (3) 1 mm; we have used the method of Ellman 181 for the assay Table 2. Kinetic constants obtained from product-inhibition studies nhibitor Variable substrate Type of inhibition nhibition constants Ki (slope) Ki (intercept) Rip Ki, CoA Glycine Non-competitive Succinyl - CoA Non-competitive Aminolevulinic acid Glycine Competitive 0.23 Succinyl-CoA Non-competitive mm Confirmation of the Kinetic Mechanism: Action of Substrates on the Holoenzyme Xpectrum The enzyme spectrum shows two absorption maxima due to the prosthetic group pyridoxal 5'-phosphate one at 415nm, the other at 330nm (Fig.3, curve ). With the addition of glycine, the absorption at 415nm decreased and there is a concomitant increase at 360 nm (Fig.3, curve 2). The addition of succinyl-coa after the action of glycine gives a spectrum comparable to the original. The
4 22 5-Aminolevulinic Acid Synthetase from Rhodopseudomonas spheroides Y addition of succinyl-coa alone does not change the spectrum. The addition of glycine after a limiting amount of succinyl-coa gives a spectrum with one absorption maximum at 415 nm and one at 360 nm (Fig. 3, curve 3). These results are consistent with the order of fixation of the substrates on the enzyme obtained a, c?? t Wavelength (nm) Fig.3. Absorption spectra of the enzyme in the presence of substrate. Addition : (1) none or 0.2 mm succinyl CoA alone or 0.2 M glycine mm succinyl-coa; (2) 0.2 M glycine; (3) 0.02 mm succinyl-coa M glycine by the previous kinetic analysis: glycine is the first substrate. Addition of CoA causes no spectral change. 5-Aminolevulinic acid gives a spectrum with an absorption maximum at 415 nm and one at 360 nm which confirms the competitive inhibition with glycine. After fixation of pyridoxal 5'-phosphate to the enzyme, there is a transimination between pyridoxal 5'-phosphate and the first substrate (glycine); succinyl-coa reacts on the complex E pyridoxal- 5'-phosphate - glycine. An enzyme * glycine - SUCcinyl-CoA ternary complex is necessarily formed in order to allow the reaction between glycine and SUCcinyl-CoA. Mechanism of nhibition by High-Energy Compounds : Analysis of Kinetic Mechanism of nhibition The effect of increasing concentrations of ATP, pyrophosphate, ADP, AMP, 3' : 5'-monophosphate, GTP and GDP on the initial rate of the reaction have been studied. Fig.4 shows the effect of ATP in double-reciprocal plot, when glycine or succinyl CoA is the varying substrate. We obtained the same curves for GTP and sodium pyrophosphate. ADP, AMP and GDP have no effect. Only ATP, GTP and pyrophosphate inhibit 5-aminolevulinic acid synthetase activity competitively with glycine and non-competitively with succinyl-coa. Plots of the intercepts and slopes which are linear functions of the reciprocal substrate concentrations, allow one to determine the inhibition constants (Table 3). Fixation of ATP and Pyrophosphate to the Enzyme Pure 5-aminolevulinic acid synthetase (fraction and fraction 11) was incubated with [N-~~PATP, t [Glycine] (rnm'') Fig.4. nhibition by ATE' of 5-aminolevulinic-acid synthdase reaction. (A) Glycine is the variable substrate and succinyl- CoA is held constant at 0.2 mm; t.he concentrations of ATP were: (1) none, (2) 0.1 mm, (3) 0.5 mm, (4) 1 mm, (5) 10 mm. [Succinyl -Con] (pm-') (B) Succinyl-CoA is the variable substrate and glycine is held constant at 0.2 M; the concentrations of ATP were: (1) none, (2) 0.1 mm, (3) 1 mm, (4) 10 mm. 1 U = 1 pmol 5-aminolevulinic acid formedx h-1
5 M. Fanica-Gaignier and J. Clement-Metral 23 Table 3. nhibition of 5-aminolevulinic acid synthetase nhibitor Variable substrate nhibition nhibition constant Ki (slope) Ki (intercept) ATP GTP Pyrophosphate GDP ADP AMP 3':5'-AMP Glycine Succinyl-CoA Glycine Succinyl-CoA Glycine Succiny 1-CoA No inhibition Competitive Non-competitive Competitive Non-compctitive Competitive Non-competitive Table 4. Estimation of --XH groups after ATP or pyrophosphate fixation to the protein Enzyme Bound ATP Free -SH groups Free -SH groups after fixation of nhibition of inhibitor enzyme activity mol/mol enzyme before after before after denaturation denaturation & f f j, & * "0 [y-32p]atp and [32P2]pyrophosphate. The incubation yielded a complex isolated by chromatography containing 1 mole of [32P]ATP or sodium [32P,]pyrophosphate per mole of enzyme. As the incubation with [LX-~~P]ATP or [y-32p]- ATP triphosphate gives a radioactive 32P * enzyme complex, we cannot eliminate the hypothesis that ATP is entirely bound to the enzyme. Experiments with [14C]ATP are necessary to confirm this hypothesis. The estimation of -SH groups [8] after ATP and pyrophosphate fixation to the protein shows that the inhibitor masks one -SH group. This is true for the two enzymes ( and 11) native and denatured (Table 4). The complexes ATP * enzyme or pyrophosphate enzyme are almost completely inactive. After incubation with p-hydroxymercuribenzoate, which blocks -SH groups, no fixation of inhibitors is obtained anymore. The results suggest that an -SH group is involved in the ATP and pyrophosphate linkage with 5-aminolevulinic acid synthetase. DSCUSSON From kinetic and spectral analysis we can propose the following sequence mechanism : Only triphosphonucleotides or pyrophosphate inhibit 5-aminolevulinic acid synthetase activity. The Ki values and the type of inhibition (competitive with glycine) suggest that t,hese inhibitors act in the same way. The -SH group of the active center is involved. An -SH group is indeed involved in the active center [9] in the substituted aldamine formed in the binding of pyridoxal 5'-phosphate to the enzyme. The site of fixation of glycine must be in the neighbourhood of this -SH group because of the transimination between pyridoxal 5'-phosphate and glycine. Now ATP is competitive with glycine and its fixation on the enzyme masks one -SH group (this is clearly demonstrated with fraction and very likely with fraction 11). A conformational change induced by ATP or pyrophosphate fixation could occur, but as denaturation by 8M urea or dodecylsulfate does not free the -SH group the conformation change is not likely be the only cause of the disappearance of the -SH group. The -SH group seems to be directly involved in ATP fixation. The very site of fixation and the type of bond remains to be determined. We are grateful to Dr G. N. Cohen for his interest in this work. Thanks are due to Mrs A. Ragnideau for her skilful technical assistance. This work constitutes a part of Doctom1 E Pyridoxal 5'-phosphate Glycine Succinyl-CoA CoASH 5-Aminolevulinic acid T L E Pyridoxal 5'-phosphate
6 24 M. Fanica-Gaignier and J. Clement-Metral : 5-Aminolevulinic Acid Synthetase from Rhodopseudoinonas spheroides Y Thesis (Doctorat 2s-Sciences) to be submitted by one of us 6a. Fanica-Gaignier, M. & Clement-Metral, J. D. (1971) (M. F. G.) to the Facultk des Sciences de Paris. Biochem. Biophys. Res. Commuib. 44, b. Fanica-Gaignier. M. & Clement-Metral. J. D. (1973) \ Eur. J. BGchem. 40, REFERENCES Urata, G. & Granick, S. J. (1963) J. Bid. Cl~em. 238, Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, iikuchi, G., iumar, A., Talmage, P. & Shemin, P. 9. Simon, E. J. & Shemin, D. (1953) J. Am. Chem. SOC. 75, (1958) J. Biol. Chem. 233, Akhtar, M. &Jordan, P. M. (1968) Chem. Commun Cha, S. & Parks, R,. E. (1964) J. Biol. Chem. 239, Warnick, G. R. & Burham, B. F. (1971) J. Biol. Chern. 11. Das, N., Cottam, G. L. & Srere, P. A. (1971) Arch. 246, Biochem. Biophys. 143, Cleland, W. W. (1963) Biochim. Biophys. Acta, 67, Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Ran- 5a. Cleland, W. W. (1963) Biochim. Biophys. Acta, 67, 173. dall, R. J. (1951) J. Biol. Chem. 193, b. Cleland, W. W. (1963) Biochim. Biophys. Acta, 67, Bray, G. A. (1960) Anal. Biochem. 1, 229. M. Fanica-Gaignier and J. Clement-Metral, Laboratoire de Photosynthbse du C.N.R.B., F Cif-sur-Yvette, France
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