Kinetic mechanism of NADH-dependent glutamate synthase from lupin nodules.

From initial-rate studies, a partially random kinetic mechanism has been deduced for NADH-dependent glutamate synthase from lupin nodules. The mechanism involves compulsory binding of NADH as first substrate, followed by random-order binding of glutamine and 2-oxoglutarate. Patterns of inhibition by glutamate substantiate the mechanism. Dithionite was incapable of acting as an alternative reducing substrate although it is known to reduce the flavine groups of the enzyme. The implications of these results are discussed. Published rate equations for this type of mechanism were found to be unsatisfactory for this enzyme and suitable new equations are produced. These equations should have general application where the obligatory first substrate binds very tightly.     

Identité des formes soluble et solubilisée à partir des mitochondries de l'alanine α-cetoglutarate aminotransferase de Lycopersicon esculentum

Alanine 2-oxoglutarate aminotransferase, extracted by Triton X-100 from tomato mitochondria, has been purified and its main kinetic characteristics determined. This form is more unstable than the soluble enzyme. However, the chromatographic patterns, effect of pH on stability, the pH optimum, the specificity and the apparent molecular weight show that it is the same enzyme and not an isoenzyme. This identity is confirmed by the results of kinetic studies and by the inhibition data by the products of the reaction for the two forms. The kinetic results also show that the two forms reversibly catalyze the transamination reaction between alanine and 2-oxoglutarate according the Ping Pong mechanism described for the soluble enzyme.     

The purification and properties of L-histidine--2-oxoglutarate aminotransferase from Pseudomonas testosteroni.

1. Inducible L-histidine--2-oxoglutarate aminotransferase was purified some 170-fold from extracts of Pseudomonas testosteroni. 2. The preparation showed only one major component after electrophoresis on polyacrylamide gels, though additional minor bands were observed when samples concentrated on a DEAE-cellulose column were used. 3. The molecular weight of the enzyme was found to be approx. 70000 by chromatography on Sephadex G-200. 4. The purification scheme produced enzyme that was inactive in the absence of pyridoxal 5'-phosphate. 5. The equilibrium constant for the reaction L-histidine+2-oxoglutarate equilibrium imidazolylpyruvate+L-glutamate was 0.49. 6. The reaction mechanism was Ping Pong. 7. The enzyme was shown to have only low activity towards aromatic amino acids and was highly specific for 2-oxoglutarate.     

Kinetic studies of glutamate dehydrogenase. The reductive amination of 2-oxoglutarate

1. Kinetic studies of the reductive amination of 2-oxoglutarate catalysed by glutamate dehydrogenase with NADH and NADPH as coenzyme were made at pH7.0 and pH 8.0. The concentrations of both substrates and coenzymes were simultaneously varied over wide ranges. Lineweaver-Burk plots with respect to each substrate and coenzyme were linear, except that with high concentrations of 2-oxoglutarate or coenzyme inhibition occurred. There was no evidence of the negative homotropic interactions between the enzyme subunits that were revealed in previous kinetic studies of the reverse reaction. 2. The initial-rate results are shown to be inconsistent with any of the six possible compulsory-order mechanisms for this three-substrate reaction, and it is concluded that a random-order mechanism is the most likely one. On the basis of this mechanism, the dissociation constants of all the binary, ternary and quaternary complexes of the enzyme and substrates are calculated from initial-rate parameters. 3. The results are discussed in relation to those of earlier workers who concluded that the mechanism is of the compulsory-order type.     

Initial-velocity kinetics of succinoyl-coenzyme A-3-oxo acid coenzyme A-transferase from sheep kidney.

The initial-velocity kinetics of sheep kidney CoA-transferase are consistent with a Ping Pong mechanism. A KAcAc-CoA of 2.7 X 10(-5) M, KSucc-CoA of 1.6 X 10(-4) M, KSucc of 5.6 X 10(-3) M and KAcAc of 6.7 X 10(-5) M were determined by using a direct assay system that monitors the concentration of magnesium acetoacetyl-CoA enolate. However, product-inhibition kinetics of sheep kidney CoA-transferase are inconsistent with a Ping Pong mechanism. The possible involvement of separate binding sites for succinate and acetoacetate are discussed.     

Ox liver glutamate dehydrogenase. The role of lysine-126 reappraised in the light of studies of inhibition and inactivation by pyridoxal 5''-phosphate.

The time-course of inactivation of bovine liver glutamate dehydrogenase by pyridoxal 5'-phosphate was studied in the presence of varied amounts of 2-oxoglutarate or NADH. Pseudo-first-order analysis reveals that the protection by both these compounds is competitive with respect to the chemical modifier. The competition is only partial, however: saturation with either NADH or 2-oxoglutarate decreases the rate constant for inactivation to a finite minimum and not to zero. Similarly, the plot of activity at equilibrium as a function of the concentration of the protecting substrate or coenzyme reveals that neither NADH nor 2-oxoglutarate protects completely against inactivation. In initial-rate experiments, pyridoxal 5'-phosphate, used as an instantaneous inhibitor rather than a long-term inactivator, displayed non-competitive inhibition with respect to both 2-oxoglutarate and NADH. These results clearly indicate that, although there is mutual hindrance between the binding to the enzyme of pyridoxal 5'-phosphate, on the one hand, and 2-oxoglutarate or NADH on the other, binding is not mutually exclusive. These findings are discussed in terms of the two-step mechanism for inactivation by pyridoxal 5'-phosphate. It is concluded that lysine-126 cannot be solely responsible for binding either the substrate or the coenzyme, but could be essential for the catalytic step.     

Glyceraldehyde-3-phosphate dehydrogenase (NADP) from Sinapis alba: Steady state kinetics

Substrate interaction and product inhibition kinetics of the forward reaction of glyceraldehyde-3-phosphate dehydrogenase (NADP) (EC 1.2.1.13) from Sinapis alba suggest an Uni Uni Uni Bi Ping Pong mechanism (NAD(P)H on, glyceraldehyde-3-phosphate off, 1,3-diphosphoglycerate on, phosphate off, NAD(P)⁺ off) with an apparent Theorell Chance displacement between 1,3-diphosphoglycerate and phosphate. The proposed mechanism predicts the existence of stable enzyme-NAD(P)⁺ and acyl-enzyme complexes as obligatory intermediates. A comparison of the present findings on the NADP-enzyme with an earlier kinetic analysis of the NAD-specific enzyme from plants (EC 1.2.1.12) by other authors shows that the kinetic mechanisms for the two enzymes, although similar in principle (both show Ping Pong kinetics), differ in some details.     

A product-inhibition study of bovine liver glutamate dehydrogenase.

1. Initial rates of oxidative deamination of L-glutamate with NAD+ as coenzyme, and of reductive aminiation of 2-oxoglutarate with NADH as coenzyme, catalysed by bovine liver glutamate dehydrogenase were measured in 0.111 M-sodium phosphate buffer, pH 7, at 25 degrees C, in the absence and presence of product inhibitors. All 12 possible combinations of variable substrate and product inhibitor were used. 2. Strict competition was observed between NAD+ and NADH, and between glutamate and 2-oxoglutarate. All other inhibition patterns were clearly non-competitive, except for inhibition by NH4+ with NAD+ as variable substrate. Here the extrapolation did not permit a clear distinction between competitive and non-competitive inhibition. 3. Mutually non-competitive behaviour between glutamate and NH4+ indicates that these substrates can be bound at the active site simultaneously. 4. Primary Lineweaver-Burk plots and derived secondary plots of slopes and intercepts against inhibitor concentration were linear, with one exception: with 2-oxoglutarate as variable substrate, the replot of primary intercepts against inhibitory NAD+ concentration was curved. 5. Separate Ki values were evaluated for the effect of each product inhibitor on the individual terms in the reciprocal initial-rate equations. With this information it is possible to calculate rates for any combination of substrate concentrations within the experimental range with any concentration of a single product inhibitor. 6. The inhibition patterns are consistent with neither a simple compulsory-order mechanism nor a rapid-equilibrium random-order mechanism without modification. They can, however, be reconciled with either type of mechanism by postulating appropirate abortive complexes. Of the two compulsory sequences that have been proposed, one, that in which the order of binding is NADH, NH4+, 2-oxoglutarate, requires an implausible pattern of abortive complex-formation to account for the results. 7. On the basis of a rapid-equilibrium random-order mechanism, dissociation constants can be calculated from the Ki values. Where these can be compared with independent estimates from the kinetics of the uninhibited reaction or from direct measurements of substrate binding, the agreement is reasonable good. On balance, therefore, the results provide further support for the rapid-equilibrium random-order mechanism under these conditions.     

Steady-state and pre-steady-state kinetic analysis of Mycobacterium smegmatis cysteine ligase (MshC)

Mycobacterium tuberculosis and many other members of the Actinomycetes family produce mycothiol, i.e., 1-d-myo-inosityl-2-(N-acetyl-l-cysteinyl)amido-2-deoxy-alpha-d-glucopyranoside (MSH or AcCys-GlcN-Ins), to act against oxidative and antibiotic stress. The biosynthesis of MSH is essential for cell growth and has been proposed to proceed via a biosynthetic pathway involving four key enzymes, MshA-MshD. The MSH biosynthetic enzymes present potential targets for inhibitor design. With this as a long-term goal, we have carried out a kinetic and mechanistic characterization, using steady-state and pre-steady-state approaches, of the recombinant Mycobacterium smegmatis MshC. MshC catalyzes the ATP-dependent condensation of GlcN-Ins and cysteine to form Cys-GlcN-Ins. Initial velocity and inhibition studies show that the steady-state kinetic mechanism of MshC is a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding followed by cysteine binding, release of PPi, binding of GlcN-Ins, followed by the release of Cys-GlcN-Ins and AMP. The steady-state kinetic parameters were determined to be kcat equal to 3.15 s-1, and Km values of 1.8, 0.1, and 0.16 mM for ATP, cysteine, and GlcN-Ins, respectively. A stable bisubstrate analogue, 5'-O-[N-(l-cysteinyl)sulfamonyl]adenosine, exhibits competitive inhibition versus ATP and noncompetitive inhibition versus cysteine, with an inhibition constant of approximately 306 nM versus ATP. Single-turnover reactions of the first and second half reactions were determined using rapid-quench techniques, giving rates of approximately 9.4 and approximately 5.2 s-1, respectively, consistent with the cysteinyl adenylate being a kinetically competent intermediate in the reaction by MshC.     

Determination of the mechanism and kinetic constants for hog kidney gamma-glutamyltransferase.

The initial-velocity kinetics of hog kidney gamma-glutamyltransferase were studied. Glutamate gamma-(4-nitroanilide) and its 3-carboxy derivative, glutamate gamma-(3-carboxy-4-nitroanilide), served as gamma-glutamyl donors, and glycylglycine as an acceptor. Reaction products were identified by paper chromatography and amino acid analysis. Inhibited Ping Pong mechanisms and a comprehensive initial- velocity expression were developed which account for the observed simultaneous gamma-glutamyl transfer and autotransfer, competitive inhibition by glycylglycine, and non-competitive inhibition by the carboxy donor. The validity of the proposed Ping Pong mechanisms are supported by enzyme-velocity data obtained with constant ratios of acceptor to donor concentrations. Kinetic constants were determined by a non-linear regression analysis. With glutamate gamma-(4-nitroanilide) as the donor, Michaelis constants for the donor, acceptor and donor-acting-as-acceptor are 1.87, 24.9, and 2.08 mM respectively. With glutamate gamma-(3-carboxy-4-nitroanilide) as the donor, these Michaelis constants are 1.63, 16.6, and 12.3 mM. Glyclyglycine competitive inhibition constants with the parent donor and its carboxy derivative are 275 and 205 mM respectively; the non-competitive inhibition constant of the carboxy donor is 34 mM.     

A kinetic study of the alpha-keto acid dehydrogenase complexes from pig heart mitochondria.

The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25 degrees. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are: 2-oxoglutarate, 0.220 mM; CoA, 0.025 mM; NAD+, 0.050 mM. Those of the pyruvate dehydrogenase complex are: pyruvate, 0.015 mM; CoA, 0.021 mM; NAD+, 0.079 mM. Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.     

Kinetic studies of the phenol sulphate-phenol sulphotransferase of Aspergillus oryzae.

Arylsulphatase II of Aspergillus oryzae exhibits both hydrolytic and sulphotransferase activities. The kinetic data suggest the formation of an intermediate covalent enzyme-sulphate complex with transfer of sulphate from donor to acceptor proceeding via a Ping Pong mechanism. The unusual kinetic behaviour when 2-hydroxy-5-nitrophenyl sulphate is the substrate is also consistent with this mechanism.     

Kinetic studies of dogfish liver glutamate dehydrogenase.

Initial-rate studies were made of the oxidation of L-glutamate by NAD+ and NADP+ catalysed by highly purified preparations of dogfish liver glutamate dehydrogenase. With NAD+ as coenzyme the kinetics show the same features of coenzyme activation as seen with the bovine liver enzyme [Engel & Dalziel (1969) Biochem. J. 115, 621--631]. With NADP+ as coenzyme, initial rates are much slower than with NAD+, and Lineweaver--Burk plots are linear over extended ranges of substrate and coenzyme concentration. Stopped-flow studies with NADP+ as coenzyme give no evidence for the accumulation of significant concentrations of NADPH-containing complexes with the enzyme in the steady state. Protection studies against inactivation by pyridoxal 5'-phosphate indicate that NAD+ and NADP+ give the same degree of protection in the presence of sodium glutarate. The results are used to deduce information about the mechanism of glutamate oxidation by the enzyme. Initial-rate studies of the reductive amination of 2-oxoglutarate by NADH and NADPH catalysed by dogfish liver glutamate dehydrogenase showed that the kinetic features of the reaction are very similar with both coenzymes, but reactions with NADH are much faster. The data show that a number of possible mechanisms for the reaction may be discarded, including the compulsory mechanism (previously proposed for the enzyme) in which the sequence of binding is NAD(P)H, NH4+ and 2-oxoglutarate. The kinetic data suggest either a rapid-equilibrium random mechanism or the compulsory mechanism with the binding sequence NH4+, NAD(P)H, 2-oxoglutarate. However, binding studies and protection studies indicate that coenzyme and 2-oxoglutarate do bind to the free enzyme.     

胆碱脱氢酶的动力学性质

本文对增溶胆碱脱氢酶的稳态初速度及产物抑制动力学做了。底物胆碱和ＰＭＳ的相互影响：变化一个底物的浓度,另一个底物的Ｋｍ及Ｖｍａｘ均变化。该酶的产物三甲胺 地 制表现为对底物胆碱非竞争性而地ＰＭＳ竞争性,在胆碱饱和的情况下,三甲胺乙醛对酶的抑制仍表现为对ＰＭＳ竞争性。这些结果表明增溶胆碱脱氢酶的催化机制搂双底物双产物乒乓机制。１－ＰＣ与９－ＡＣ对增溶胆碱脱氢酶均有抑制作用,且均为混和型抑制,Ｋ１分别     

A steady-state random-order mechanism for the oxidative deamination of norvaline by glutamate dehydrogenase.

The kinetic mechanism of glutamate dehydrogenase with the monocarboxylic substrate norvaline was examined by using initial-rate steady-state kinetics and inhibition kinetics. To a first approximation the reaction mechanism can be described as a rapid-equilibrium random-order one. Binding synergism between the monocarboxylic substrate and coenzyme is not observed. Dissociation constants for NAD+ and 2-oxoglutarate calculated from the kinetic data assuming a rapid-equilibrium random-order model are in good agreement with independently obtained estimates. Lineweaver-Burk plots with varied norvaline concentration are not strictly linear, and it is concluded that a steady-state random-order model more accurately reflects the observed kinetics with norvaline as substrate.     

Kinetic studies of the fatty acid synthetase multienzyme complex from Euglena gracilis variety bacillaris.

A fatty acid synthetase multienzyme complex was purified from Euglena gracilis variety bacillaris. The fatty acid synthetase activity is specifically inhibited by antibodies against Escherichia coli acyl-carrier protein. The Euglena enzyme system requires both NADPH and NADH for maximal activity. An analysis was done of the steady-state kinetics of the reaction catalysed by the fatty acid synthetase multienzyme complex. Initial-velocity studies were done in which the concentrations of the following pairs of substrates were varied: malonyl-CoA and acetyl-CoA, NADPH and acetyl-CoA, malonyl-CoA and NADPH. In all three cases patterns of the Ping Pong type were obtained. Product-inhibition studies were done with NADP+ and CoA. NADP+ is a competitive inhibitor with respect to NADPH, and uncompetitive with respect to malonyl-CoA and acetyl-CoA. CoA is uncompetitive with respect to NADPH and competitive with respect to malonyl-CoA and acetyl-CoA. When the concentrations of acetyl-CoA and malonyl-CoA were varied over a wide range, mutual competitive substrate inhibition was observed. When the fatty acid synthetase was incubated with radiolabelled acetyl-CoA or malonyl-CoA, labelled acyl-enzyme was isolated. The results are consistent with the idea that fatty acid synthesis proceeds by a multisite substituted-enzyme mechanism involving Ping Pong reactions at the following enzyme sites: acetyl transacylase, malonyl transacylase, beta-oxo acyl-enzyme synthetase and fatty acyl transacylase.     

Isoleucyl transfer ribonucleic acid synthetase. Steady-state kinetic analysis.

A steady-state kinetic analysis was conducted of the overall aminoacylation reaction catalyzed by isoleucyl-tRNA synthetase. The patterns of Lineweaver-Burk plots obtained indicated that tRNA adds to the enzyme only after isoleucyl adenylate formation and pyrophosphate release. These kinetic patterns were consistent with the bi-uni-uni-bi Ping Pong mechanism generally accepted for this aminoacyl-tRNA synthetase, but they could also be accommodated by a mechanism in which a second molecule of L-isoleucine added to the enzyme between isoleucyl adenylate formation and aminoacylation of tRNA [Fersht, A.R., & Kaethner, M.M. (1976) Biochemistry 15, 818]. The values of the kinetic parameters favor the latter mechanism. The results of this kinetic analysis indicated that the affinity of isoleucyl-tRNA synthetase for Mg.ATP was enhanced upon binding of L-isoleucine and vice versa. It also indicated that the affinity of the enzyme for L-isoleucine is decreased upon binding tRNA and vice versa. The values of dissociation constants calculated for each of the substrates by this study generally compared well with those determined by other authors using a variety of kinetic and equilibrium methods.     

Rat liver mitochondrial monoamine oxidase. A change in the reaction mechanism on solubilization.

1. The kinetics of benzylamine oxidation by a soluble preparation of rat liver mitochondrial monoamine oxidase were investigated and were shown to conform to adouble-displacement (or Ping Pong) mechanism. 2. The pathway differs in detail from that followed by other amine oxidases, including the membrane-bound enzyme in rat liver mitochondrial outer membranes. 3. It is suggested taht the conformation of the protein in the soluble state differs from that in the membrane-bound state. 4. The full rate equations for this mechanism have been deposited as Supplementary Publication SUP 50039 (5pages) at the British Library (lending Division) (formely the National Lending Library for Science and Technology), Boston Spa, Yorks, LS237BQ, U.K.. from whom copies can be obtained on the terms indicated in Biochem. J (1975) 145,5.     

Kinetic studies with the low-Km aldehyde reductase from ox brain.

Initial-rate studies of the low-Km aldehyde reductase-catalysed reduction of pyridine-3-aldehyde by NADPH gave families of parallel double-reciprocal plots, consistent with a double-displacement mechanism being obeyed. Studies on the variation of the initial velocity with the concentration of a mixture of the two substrates were also consistent with a double-displacement mechanism. In contrast, the initial-rate data indicated that a sequential mechanism was followed when NADH was used as the coenzyme. Product-inhibition studies, however, indicated that a compulsory-order mechanism was followed in which NADPH bound before pyridine-3-aldehyde with a ternary complex being formed and the release of pyrid-3-ylcarbinol before NADP+. The apparently parallel double-reciprocal plots obtained in the initial-rate studies with NADPH and pyridine-3-aldehyde were thus attributed to the apparent dissociation constant for the binary complex between the enzyme and coenzyme being finite but very low.