Kinetics of human erythrocyte glucose-6-phosphate dehydrogenase dimers

The steady-state kinetics of human erythrocyte glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) dimers were studied by initial rate measurement. These experiments gave intersecting double-reciprocal plots suggesting a ternary complex mechanism with a Km for NADP and glucose 6-phosphate of 11 microM and 43 microM, respectively. These studies were combined with rate measurements in the presence of one product (NADPH), dead-end inhibitors, as well as alternative substrates. The inhibition by NADPH was found to be competitive with respect to both substrates. Alternate substrates experiments gave linear double-reciprocal plots over a wide range of substrate concentrations. The results suggest that the dimeric enzyme follows either a random or a Theorell-Chance mechanism.     

Properties of rat heart adenosine kinase.

Adenosine kinase was purified 870-fold from rat heart by a combination of gel filtration and affinity chromatography. The preparation was free of purine-metabolizing enzymes that could interfere in the assay of the kinase. A study of the properties of the purified enzyme showed that it is activated by Na+ and K+, it possesses a broad pH optimum between 6 and 8, MgATP is the nucleotide substrate, free Mg2+ is an inhibitor with respect to both MgATP and adenosine, and the enzyme is subject to substrate inhibition by adenosine. The severity of this inhibition increases as the concentration of free Mg2+ increase. The Km for MgATP was calculated to be 0.8 mM and that for adenosine, at likely physiological concentrations of MgATP and free MgCl2, was about 0.2 microM. In vivo the enzyme is likely to be saturated with both MgATP and adenosine. Indeed, the adenosine concentration in rat heart in vivo is probably sufficient to cause substrate inhibition, and this would be increased by an increase in free Mg2+ concentration. Changes in the concentrations of adenosine and free Mg2+ may play a role in modifying the activity of the enzyme in vivo.     

Regulation of smooth-muscle myosin-light-chain kinase. Steady-state kinetic studies of the reaction mechanism

The kinetic mechanism of turkey gizzard smooth muscle myosin-light-chain kinase was investigated using the isolated 20-kDa light chain of myosin as substrate. The kinetic and product inhibition patterns of the forward reaction indicated an ordered sequential mechanism in which MgATP bound first, ADP was released last. The order of substrate binding and product release was confirmed independently by competitive, dead-end inhibition patterns obtained using the non-hydrolizable ATP analog adenosine 5'-[beta,gamma-imido]triphosphate. The mechanism was also characterized by a relatively strong product inhibition by ADP and a weak one by phosphorylated 20-kDa light-chain myosin, in addition to a significant inhibition by the latter product via a formation of a dead-end complex. [gamma-32P]ATP in equilibrium with [32P]phosphorylated light chain isotope-exchange data were consistent with the deduced mechanism and with the presence of the latter dead-end complex.     

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. III. A minimal model

A steady-state kinetic investigation of the effect of K+ on the Na+-enzyme activity of the (Na+ + K+)-ATPase in broken membrane preparations is reported. Analysis of the kinetic patterns obtained, together with the results reported in the first two articles of this series permit the following conclusions. 1. K+ inhibits the Na+-enzyme (the enzyme activity measured at micromolar substrate concentrations in the presence of Na+). The inhibition of non-competitive at low and competitive at higher K+ concentrations and is enhanced by free Mg2+. 2. The results indicate that the Na+-enzyme at steady-state tends to be accumulated in an enzyme-potassium complex when K+ is added. 3. The enzyme-potassium complex, in turn, binds Mg2+ in a dead-end fashion. The dissociation constant for the enzyme-K-Mg complex, estimated from the data, is 7.2 mM. The same value was obtained earlier for the Mg2+ inhibition constant of the substrate-free form of the (Na+ + K+)-enzyme (the enzyme activity measured with Na+ and K+ and at millimolar substrate concentrations) suggesting that the two constants describe the same equilibrium. 4. On the basis of the known (optimal) activity of the (Na+ + K+)-ATPase, relative to that of the Na+-ATPase, a rate constant condition is found which must be met if the Post-Albers kinetic scheme is to satisfy the data. Kinetic data for the phosphoenzyme indicate that this condition is not satisfied. 5. On the basis of the kinetic results a model for the hydrolytic action of (Na+ + K+)-ATPase is proposed. This model encompasses the Post-Albers scheme but contains two distinctive hydrolysis cycles (an 'Na+-enzyme cycle' and a '(Na+ + K+)-enzyme cycle') with widely different affinities for the substrates. Only one of the cycles (the Na+-enzyme cycle) involves acid-stable phosphorylated enzyme intermediates at discernible steady-state concentrations. Which of the two main cycles is predominant in any particular system is determined by the concentration of ligands and substrates. 6. According to this scheme, an enzyme preparation may exhibit both a high (Na+-enzyme) and a low ((Na+ + K+)-enzyme) substrate affinity, without the necessity of assigning more than one substrate site to a particular enzyme unit at any one time.     

Random-order ternary complex reaction mechanism of serine acetyltransferase from Escherichia coli

Although serine acetyltransferase (SAT) from Escherichia coli is homologous with a number of bacterial enzymes that catalyze O-acetyl transfer by a sequential (ternary complex) mechanism, it has been suggested, from experiments with the nearly identical enzyme from Salmonella typhimurium, that the reaction could proceed via an acetyl-enzyme intermediate. To resolve the matter, the E. coli gene for SAT was overexpressed and the enzyme purified 13-fold to homogeneity. The results of a steady-state kinetic analysis of the forward reaction are diagnostic for a ternary complex mechanism, and the response of SAT to dead-end inhibitors indicates a random order for the addition of substrates. The linearity of primary double-reciprocal plots, in the presence and absence of dead-end inhibitors, argues that interconversion of ternary complexes is not significantly faster than kcat, whereas substrate inhibition by serine suggests that breakdown of the SAT.CoA binary complex is rate-determining. The results of equilibrium isotope exchange experiments, for both half-reactions, rule out a "ping-pong" mechanism involving an acetyl-enzyme intermediate, and a pre-steady-state kinetic analysis of the turnover of AcCoA supports such a conclusion. Kinetic data for the reverse reaction (acetylation of CoA by O-acetylserine) are also consistent with a steady-state random-order mechanism, wherein both the breakdown of the SAT*serine complex and the interconversion of ternary complexes are partially rate-determining.     

D-Mannitol dehydrogenase from Absidia glauca. Steady-state kinetic properties and the inhibitory role of mannitol 1-phosphate.

Steady-state kinetic studies including initial velocity for mannitol oxidation and fructose reduction and product inhibition for mannitol oxidation using fructose and reduced nicotinamide adenine dinucleotide (NADH) are in accord with a reaction mechanism best described as ordered Bi-Bi with NAD+ and NADH designated as the first substrate, last product, respectively at pH 8.8. All replots of slopes and intercepts from product inhibition studies were linear. Dead-end inhibition studies using mannitol 1-phosphate gave slope-parabolic, intercept-linear noncompetitive inhibition for both NAD+ and mannitol as substrates. The dead-end inhibitor is capable of binding multiply to the E, EA, and EQ forms of the enzyme to an extent that is controlled by the concentration of substrates. The EQ complex is inferred to undergo a conformational change, E'Q equilibrium EQ, since (V1/E1) greater than (KiqV2)/(KqE1), and no evidence for dead-end complex formation with NADH can be adduced. This is interpreted to mean that the release of fructose from the central complex is faster than the isomerization of the E-NADH complex. When mannitol is saturating, the noncompetitive inhibition against NAD+, as the variable substrate, becomes parabolic uncompetitive. A replot of the slopes of the parabola against mannitol 1-phosphate remains concave upward. This situation could arise if the conformational change we infer in the EQ complex opens up additional sites on the protein which can interact with the dead-end inhibitor.     

Effect of ATP on phosphofructokinase-2 from Escherichia coli. A mutant enzyme altered in the allosteric site for MgATP

The activity of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2* was measured over a wide range of Mg2+ and ATP concentrations. MgATP2- inhibited only the Pfk-2 enzyme, with a degree of cooperativity of 1.5. This inhibition was relieved upon increasing the fructose-6-P concentration or by lowering the pH of the reaction mixture. Other nucleotides used as phosphate donors instead of ATP did not inhibit. MgATP2- was the true substrate for both enzymes and their Km values for this compound were not affected by an increase of the free Mg2+ concentration. However, free Mg2+ partially relieved the MgATP2- inhibition of Pfk-2 under conditions where the ATP4- concentration was negligible, without changes in the degree of cooperativity. ATP4- acted as a strong competitive inhibitor of both Pfk-2 and Pfk-2* with respect to MgATP2- with Ki values of 10 and 8 microM, respectively. ADP, AMP, and cAMP did not prevent the MgATP2- inhibition of Pfk-2. These results suggest the presence of an allosteric site for MgATP2- in Pfk-2 responsible for the MgATP2- inhibition, which is altered in Pfk-2* as a consequence of the structural mutation.     

Substrate inhibition in a human variant of hypoxanthine-guanine phosphoribosyltransferase

Hypoxanthine-guanine phosphoribosyltransferase from a young man with purine overproduction and decreased purine salvage in fibroblast cultures was found to have low activity at concentrations of purine substrates at which the enzyme from normal individuals showed near maximal activity. The low enzyme activity was not associated with changes in the values of the Km(app) and Vmax(app) for any of the enzyme substrates. However, the enzyme activity was susceptible to substrate inhibition by hypoxanthine and guanine. The values obtained for the true Km, true Vmax, and true Ki for hypoxanthine were 26 +/- 10 microM, 1761 +/- 382 microunits/mg of protein, and 80 +/- 20 microM, respectively. The pattern of the substrate inhibition, as seen on a plot of 1/v versus hypoxanthine concentration, was characteristic of that associated with the formation of a dead-end complex between the inhibitory substrate and an enzyme form with which it normally does not react. The nature of this enzyme form and that of the dead-end complex was determined from double inhibition experiments, which indicated that hypoxanthine interacted with an enzyme-PPi intermediate to form an enzyme-hypoxanthine-PPi dead-end complex. The trapping of the enzyme in this inactive form explains the low activity at high purine base concentrations. Further information as to the nature of the reaction mechanism was obtained from plots of the reciprocal of enzyme activity versus the reciprocal of PP-ribose-P concentration at different fixed hypoxanthine concentrations. A pattern characteristic of uncompetitive substrate inhibition was obtained. This is indicative of an ordered sequential binding of substrates on the enzyme; PP-ribose-P binding before hypoxanthine. Thus, the variant enzyme showed an ordered sequential reaction mechanism, with the inhibitory substrate forming a dead-end complex with an enzyme-PPi intermediate.     

Inhibition of skeletal muscle sarcoplasmic reticulum CaATPase activity by calmidazolium

Calmidazolium, a lipophilic cation and putative calmodulin-specific antagonist, inhibited potently the calcium ATPase of sarcoplasmic reticulum (SR) vesicles isolated from skeletal muscle. Based on steady-state measurements of catalytic activity over a range of MgATP, calmidazolium, and SR protein concentrations, the calculated values of the inhibition constant (KI) and binding stoichiometry were 0.06 microM and 770 nmol/mg protein, respectively. SR CaATPase inhibition apparently is not a general property of lipophilic cations since the hydrophobic anion tetraphenylboron inhibited catalysis, whereas its cationic analog, tetraphenylarsonium, did not. Enzyme inhibition by calmidazolium was noncompetitive with respect to the substrates Ca2+ and MgATP. In the presence of other SR CaATPase inhibitors, calmidazolium was competitive with respect to quercetin and noncompetitive with respect to trifluoperazine and propranolol. While calmidazolium inhibited enzyme phosphorylation by MgATP, catalysis was more sensitive to the inhibitor. Binding of calmidazolium to SR membranes produced morphological changes seen by electron microscopy as membrane thickening and loss of resolution of surface detail. Our results show that calmidazolium is a high-affinity, noncompetitive inhibitor of skeletal SR CaATPase activity, and they suggest that this inhibition is based on binding to the membrane phospholipids rather than specific antagonism of enzyme activation by calmodulin.     

The mechanism and modes of inhibition of arginine kinase from the cockroach (Periplaneta americana)

The kinetic mechanism and evaluation of several potential inhibitors of purified arginine kinase from the cockroach (Periplanta americana) were investigated. This monomeric phosphagen kinase is important in maintaining ATP levels during the rapid energy demands of muscle required for contraction and motility. Analysis reveals the following dissociation constants (mM) for the binary complex: E.Arg P-->E+Arg P, K=1.0; E.Arg-->E+Arg, K=0.45; E.MgATP-->E+MgATP, K=0.17; E.MgADP-->E+MgADP, K=0.12; and the ternary complex: Arg P.E.MgADP-->E.MgADP+Arg P, K=0.94; Arg.E.MgATP-->E.MgATP+Arg, K=0.49; MgATP.Enz.Arg-->E.Arg+MgATP, K=0.14; MgADP.E.Arg P-->E.Arg P+MgADP, K=0.09. For a particular substrate, the ratio of the dissociation constants for the binary to ternary complex is close to one, indicating little, if any, cooperativity in substrate binding for the rapid equilibrium, random addition mechanism. The time course of the arginine kinase reaction exhibits a pronounced curvature, which, as described for enzyme from other sources, is attributed to formation of an inhibitory catalytic dead-end complex, MgADP.E.Arg. The curvature is accentuated by the addition of monovalent anions, including borate, thiocyanate, and, most notably, nitrite and nitrate. This effect is attributed to stabilization of the dead-end complex through formation of a transition state analog. However, the substantial decrease in initial velocity (92%) caused by nitrate is due to an additional inhibitory effect, further characterized as non-competitive inhibition (Ki=8.0 mM) with the substrate L-arginine. On the other hand, borate inhibition of the initial velocity is only 30% with significant subsequent curvature, suggesting that this anion functions as an inhibitor mainly by formation of a transition state analog. However, some component of the borate inhibition appears to be mediated by an apparent partial competitive inhibition with L-arginine. D-arginine is not a substrate for arginine kinase from the cockroach, but is an effective competitive inhibitor with a Ki=0.31 mM. L-Canavanine is a weak substrate for arginine kinase (Km=6.7 mM) with a Vmax for the pure enzyme that is approximately one-third that of L-arginine. However, initial velocity experiments of substrate mixtures suggest that competition between L-canavanine and L-arginine may not be a simple summation effect and may involve a structural modification. Sensitivity of arginine kinase activity to D-arginine as well as nitrate and borate anions, coupled with the fact that L-arginine is an essential amino acid for the cockroach, suggest that arginine kinase could be a useful chemotherapeutic target for the control of cockroach proliferation.     

Kinetic studies on the reaction catalysed by phosphofructokinase from Trypanosoma brucei.

The steady-state kinetics of the reaction catalysed by the bloodstream form of Trypanosoma brucei were studied at pH 6.7. In the presence of 50 mM-potassium phosphate buffer, the apparent co-operativity with respect to fructose 6-phosphate and the non-linear relationship between initial velocity and enzyme concentration, which were found when the enzyme was assayed in 50 mM-imidazole buffer [Cronin & Tipton (1985) Biochem. J. 227, 113-124], are not evident. Studies on the variations of the initial rate with changing concentrations of MgATP and fructose 6-phosphate, the product inhibition by fructose 1,6-bisphosphate and the effects of the alternative substrate ITP were consistent with an ordered reaction pathway, in which MgATP binds to the enzyme before fructose 6-phosphate, and fructose 1,6-bisphosphate is the first product to dissociate from the ternary complex.     

Mechanism of ketol acid reductoisomerase--steady-state analysis and metal ion requirement

Ketol acid reductoisomerase is an enzyme of the branched-chain amino acid biosynthetic pathway. It catalyzes two separate reactions: an acetoin rearrangement and a reduction. This paper reports on the purification of the enzyme from a recombinant Escherichia coli and on the steady-state kinetics of the enzyme. The kinetics of the reaction were determined for the forward and reverse reaction by using the appropriate chiral substrates. At saturating metal ion concentrations the mechanism follows an ordered pathway where NADPH binds before acetolactate. The product of the rearrangement of acetolactate, 3-hydroxy-3-methyl-2-oxobutyrate, is shown to be kinetically competent as an intermediate in the enzyme-catalyzed reaction. Starting with acetolactate, Mg2+ is the only divalent metal ion that will support enzyme catalysis. For the reduction of 3-hydroxy-3-methyl-2-oxobutyrate, Mn2+ is catalytically active. Product and dead-end inhibition studies indicate that the binding of metal ion and NADPH occurs randomly. In the forward reaction direction, the deuterium kinetic isotope effect on V/K is 1.07 when acetolactate is the substrate and 1.39 when 3-hydroxy-3-methyl-2-oxobutyrate is the substrate.     

Steady-state kinetic mechanism of Ras farnesyl:protein transferase.

The steady-state kinetic mechanism of bovine brain farnesyl:protein transferase (FPTase) has been determined using a series of initial velocity studies, including both dead-end substrate and product inhibitor experiments. Reciprocal plots of the initial velocity data intersected on the 1/[s] axis, indicating that a ternary complex forms (sequential mechanism) and suggesting that the binding of one substrate does not affect the binding of the other. The order of substrate addition was probed by determining the patterns of dead-end substrate and product inhibition. Two nonhydrolyzable analogues of farnesyl diphosphate, (alpha-hydroxyfarnesyl)phosphonic acid (1) and [[(farnesylmethyl)hydroxyphosphinyl]methyl]phosphonic acid (2), were both shown to be competitive inhibitors of farnesyl diphosphate and noncompetitive inhibitors of Ras-CVLS. Four nonsubstrate tetrapeptides, CV[D-L]S, CVLS-NH2, N-acetyl-L-penicillamine-VIM, and CIFM, were all shown to be noncompetitive inhibitors of farnesyl diphosphate and competitive inhibitors of Ras-CVLS. These data are consistent with random order of substrate addition. Product inhibition patterns corroborated the results found with the dead-end substrate inhibitors. We conclude that bovine brain FPTase proceeds through a random order sequential mechanism. Determination of steady-state parameters for several physiological Ras-CaaX variants showed that amino acid changes affected the values of KM, but not those of kcat, suggesting that the catalytic efficiencies (kcat/KM) of Ras-CaaX substrates depend largely upon their relative binding affinity for FPTase.     

The roles of magnesium ions in the reaction catalysed by phosphofructokinase from Trypanosoma brucei.

The involvement of Mg2+ ions in the reaction catalysed by phosphofructokinase from Trypanosoma brucei was studied. The true substrate for the enzyme was shown to be the MgATP2-complex, and free Mg2+ ions are also required for enzyme activity. At concentrations of MgATP2- of 2.92 mM and greater, and a fructose 6-phosphate concentration of 1 mM and in the presence of EDTA as a Mg2+ buffer, the Km value for Mg2+ was determined to be 294 +/- 18 microM. Neither MgATP nor free ATP is an inhibitor of the enzyme, although apparent inhibition by the latter can be observed as a consequence of the decrease in free Mg2+ by chelation.     

Complete kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae

The kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae was determined using initial velocity studies in the absence and presence of product and dead end inhibitors in both reaction directions. Data suggest a steady state random kinetic mechanism. The dissociation constant of the Mg-homoisocitrate complex (MgHIc) was estimated to be 11 +/- 2 mM as measured using Mg2+ as a shift reagent. Initial velocity data indicate the MgHIc complex is the reactant in the direction of oxidative decarboxylation, while in the reverse reaction direction, the enzyme likely binds uncomplexed Mg2+ and alpha-ketoadipate. Curvature is observed in the double-reciprocal plots for product inhibition by NADH and the dead-end inhibition by 3-acetylpyridine adenine dinucleotide phosphate when MgHIc is the varied substrate. At low concentrations of MgHIc, the inhibition by both nucleotides is competitive, but as the MgHIc concentration increases, the inhibition changes to uncompetitive, consistent with a steady state random mechanism with preferred binding of MgHIc before NAD. Release of product is preferred and ordered with respect to CO2, alpha-ketoadipate, and NADH. Isocitrate is a slow substrate with a rate (V/E(t)) 216-fold slower than that measured with HIc. In contrast to HIc, the uncomplexed form of isocitrate and Mg2+ bind to the enzyme. The kinetic mechanism in the direction of oxidative decarboxylation of isocitrate, on the basis of initial velocity studies in the absence and presence of dead-end inhibitors, suggests random addition of NAD and isocitrate with Mg2+ binding before isocitrate in rapid equilibrium, and the mechanism approximates rapid equilibrium random. The Keq for the overall reaction measured directly using the change in NADH as a probe is 0.45 M.     

A kinetic study of rabbit muscle pyruvate kinase

The paper reports a study of the kinetics of the reaction between phosphoenolpyruvate, ADP and Mg(2+) catalysed by rabbit muscle pyruvate kinase. The experimental results indicate that the reaction mechanism is equilibrium random-order in type, that the substrates and products are phosphoenolpyruvate, ADP, Mg(2+), pyruvate and MgATP, and that dead-end complexes, between pyruvate, ADP and Mg(2+), form randomly and exist in equilibrium with themselves and other substrate complexes. Values were determined for the Michaelis, dissociation and inhibition constants of the reaction and are compared with values ascertained by previous workers.     

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. I. Substrate identity

A detailed steady-state kinetic investigation of the hydrolysis of ATP catalyzed by (Na+ + K+)-ATPase is reported. The activity was studied in the presence of (i) Na+ (130 mM), K+ (20 mM) and micromolar ATP concentrations and Na+ (150 mM) the ('Na+-enzyme'). The data obtained lead to the following results: 1. The action of each enzyme may be described by a simple kinetic mechanism with one (Na+-enzyme) or two ((Na+ + K+)-enzyme) dead-end Mg complexes. 2. For both enzymes, both MgATP and free ATP are substrates, with Mg2+, in the latter case, as the second substrate. 3. For each enzyme, the complete set of kinetic constants (seven for the Na+-enzyme, eight for the (Na+ + K+)-enzyme) are determined from the data. 4. For each enzyme it is shown that, in the alternate substrate mechanism obtained, the ratio of net steady-state flux along the 'MgATP pathway' to that of the 'ATP-Mg pathway' increases linearly with the concentration of free Mg2+. The parameters of this function are determined from the data. As a result of this, at high (greater than 3 mM) free Mg2+ concentrations the alternate substrate mechanism degenerates into a 'limiting' kinetic mechanism, with MgATP as the (essentially) sole substrate, and Mg2+ as an uncompetitive (Na+-enzyme) or non-competitive ((Na+ + K+)-enzyme) inhibitor.     

Kinetic characterization of the calmodulin-activated catalytic subunit of phosphorylase kinase

The phosphorylase kinase holoenzyme from skeletal muscle is composed of a catalytic and three different regulatory subunits. Analysis of the kinetic mechanism of the holoenzyme is complicated because both the natural substrate phosphorylase b and also phosphorylase kinase itself have allosteric binding sites for adenine nucleotides. In the case of the kinase, these allosteric sites are not on the catalytic subunit. We have investigated the kinetic mechanism of phosphorylase kinase by using its isolated catalytic gamma-subunit (activated by calmodulin) and an alternative peptide substrate (SDQEKRKQISVRGL) corresponding to the convertible region of phosphorylase b, thus eliminating from our system all known allosteric binding sites for nucleotides. This peptide has been previously employed to study the kinetic mechanism of the kinase holoenzyme before the existence of the allosteric sites on the regulatory subunits was suspected [Tabatabai, L. B., & Graves, D. J. (1978) J. Biol. Chem. 253, 2196-2202]. This peptide was determined to be as good an alternative substrate for the isolated catalytic subunit as it was for the holoenzyme. Initial velocity data indicated a sequential kinetic mechanism with apparent Km's for MgATP and peptide of 0.07 and 0.47 mM, respectively. MgADP used as product inhibitor showed competitive inhibition against MgATP and noncompetitive inhibition against peptide, whereas with phosphopeptide as product inhibitor, the inhibition was competitive against both MgATP and peptide. The initial velocity and product inhibition studies were consistent with a rapid equilibrium random mechanism with one abortive complex, enzyme-MgADP-peptide. The substrate-directed, dead-end inhibitors 5'-adenylyl imidodiphosphate and Asp-peptide, in which the convertible Ser of the alternative peptide substrate was replaced with Asp, were competitive inhibitors toward their like substrates and noncompetitive inhibitors toward their unlike substrates, further supporting a random mechanism, which was also the conclusion from the report cited above that used the holoenzyme.     

The kinetic mechanisms of the bifunctional enzyme aspartokinase-homoserine dehydrogenase I from Escherichia coli

The kinetic mechanisms of the reactions catalyzed by the two catalytic domains of aspartokinase-homoserine dehydrogenase I from Escherichia coli have been determined. Initial velocity, product inhibition, and dead-end inhibition studies of homoserine dehydrogenase are consistent with an ordered addition of NADPH and aspartate beta-semialdehyde followed by an ordered release of homoserine and NADP+. Aspartokinase I catalyzes the phosphorylation of a number of L-aspartic acid analogues and, moreover, can utilize MgdATP as a phosphoryl donor. Because of this broad substrate specificity, alternative substrate diagnostics was used to probe the kinetic mechanism of this enzyme. The kinetic patterns showed two sets of intersecting lines that are indicative of a random mechanism. Incorporation of these results with the data obtained from initial velocity, product inhibition, and dead-end inhibition studies at pH 8.0 are consistent with a random addition of L-aspartic acid and MgATP and an ordered release of MgADP and beta-aspartyl phosphate.