Enzyme-catalyzed acylation of homoserine: mechanistic characterization of the Escherichia coli metA-encoded homoserine transsuccinylase

The first unique step in bacterial and plant methionine biosynthesis involves the activation of the gamma-hydroxyl of homoserine. In Escherichia coli, this activation is accomplished via a succinylation reaction catalyzed by homoserine transsuccinylase. The activity of this enzyme is closely regulated in vivo and therefore represents a critical control point for cell growth and viability. We have cloned homoserine transsuccinylase from E. coli and present the first detailed enzymatic study of this enzyme. Steady-state kinetic experiments demonstrate that the enzyme utilizes a ping-pong kinetic mechanism in which the succinyl group of succinyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine to form the final product, O-succinylhomoserine. The maximal velocity, V/K(succinyl)(-)(CoA), and V/K(homoserine) all exhibited a bell-shaped pH dependence with apparent pK's of 6.6 and approximately 7.9. The enzyme was inhibited by iodoacetamide in a pH-dependent manner, with an apparent pK of the group being inactivated of 6.4. This suggests the presence of an active site cysteine which forms a succinyl-cysteine intermediate during enzymatic turnover. Solvent kinetic isotope effect studies yielded inverse effects of 0.7 on V and 0.61 on V/K in the reverse reaction only. On the basis of these observations, we propose a detailed chemical mechanism for this important member of the acyltransferase family.     

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.     

Isotope effect studies of the chemical mechanism of pig heart NADP isocitrate dehydrogenase

The catalytic mechanism of porcine heart NADP isocitrate dehydrogenase has been investigated by use of the variation of deuterium and 13C kinetic isotope effects with pH. The observed 13C isotope effect on V/K for isocitrate increases from 1.0028 at neutral pH to a limiting value of 1.040 at low pH. The limiting 13C isotope effect with deuteriated isocitrate at low pH is 1.016. This decrease in 13(V/KIc) upon deuteriation indicates a stepwise mechanism for the oxidation and decarboxylation of isocitrate. This predicts a deuterium isotope effect on V/K of 2.9, but D(V/K) at low pH only increases to a maximum of 1.08. It is not known why 13(V/KIc) with deuteriated isocitrate decreases more than predicted. The pK seen in the 13(V/KIc) pH profile for isocitrate is 4.5. This pK is displaced 1.2 pH units from the true pK of the acid/base functionality of 5.7 seen in the pKi profile for oxalylglycine, a competitive inhibitor for isocitrate. From this displacement, catalysis is estimated to be 16 times faster than substrate dissociation. By use of the pH-dependent partitioning ratio of the reaction intermediate oxalosuccinate between decarboxylation to 2-ketoglutarate and reduction to isocitrate, the forward commitment to catalysis for decarboxylation was determined to be 7.3 at pH 5.4 and 3.2 at pH 5.0. This gives an intrinsic 13C isotope effect for decarboxylation of 1.050. 3-Fluoroisocitrate is a new substrate oxidatively decarboxylated by NADP isocitrate dehydrogenase. At neutral pH, D(V/K3-F-Ic) = 1.45 and 13(V/K3-F-Ic) = 1.0129. At pH 5.2, 13(V/K3-F-Ic) increases to 1.0186, indicating that a finite, but diminished, external commitment remains at neutral pH. The product of oxidative decarboxylation of 3-hydroxyisocitrate by NADP isocitrate dehydrogenase is 2-hydroxy-3-ketoglutarate. This results from enzymatic protonation of the cis-enediol intermediate at C2 rather than C3 (as seen with isocitrate and 3-fluoroisocitrate). 2-Hydroxy-3-ketoglutarate further decarboxylates in solution to 2-hydroxy-3-ketobutyrate, which further decarboxylates to acetol. This makes 3-hydroxyisocitrate unsuitable for 13C isotope effect studies.     

Substrate specificity and kinetic isotope effect analysis of the Eschericia coli ketopantoate reductase

Ketopantoate reductase (EC 1.1.1.169), an enzyme in the pantothenate biosynthetic pathway, catalyzes the NADPH-dependent reduction of alpha-ketopantoate to form D-(-)-pantoate. The enzyme exhibits high specificity for ketopantoate, with V and V/K for ketopantoate being 5- and 365-fold higher than those values for alpha-ketoisovalerate and 20- and 648-fold higher than those values for alpha-keto-beta-methyl-n-valerate, respectively. For pyridine nucleotides, V/K for beta-NADPH is 3-500-fold higher than that for other nucleotide substrates. The magnitude of the primary deuterium kinetic isotope effects on V and V/K varied substantially when different ketoacid and pyridine nucleotide substrates were used. The small primary deuterium kinetic isotope effects observed using NADPH and NHDPH suggest that the chemical step is not rate-limiting, while larger primary deuterium isotope effects were observed for poor ketoacid and pyridine nucleotide substrates, indicating that the chemical reaction has become partially or completely rate-limiting. The pH dependence of (D)V using ketopantoate was observed to vary from a value of 1.1 at low pH to a value of 2.5 at high pH, while the magnitude of (D)V/K(NADPH) and (D)V/K(KP) were pH-independent. The value of (D)V is large and pH-independent when alpha-keto-beta-methyl-n-valerate was used as the ketoacid substrate. Solvent kinetic isotope effects of 2.2 and 1.2 on V and V/K, respectively, were observed with alpha-keto-beta-methyl-n-valerate. Rapid reaction analysis of NADPH oxidation using ketopantoate showed no "burst" phase, suggesting that product-release steps are not rate-limiting and the cause of the small observed kinetic isotope effects with this substrate pair. Large primary deuterium isotope effects on V and V/K using 3-APADPH in steady-state experiments, equivalent to the isotope effect observed in single turnover studies, suggests that chemistry is rate-limiting for this poorer reductant. These results are discussed in terms of a kinetic and chemical mechanism for the enzyme.     

Chemical mechanism of the adenosine cyclic 3',5'-monophosphate dependent protein kinase from pH studies

The pH dependence of kinetic parameters and inhibitor dissociation constants for the adenosine cyclic 3',5'-monophosphate dependent protein kinase reaction has been determined. Data are consistent with a mechanism in which reactants selectively bind to enzyme with the catalytic base unprotonated and an enzyme group required protonated for peptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) binding. Binding of the peptide apparently locks both of the above enzyme residues in their correct protonation state. MgATP preferentially binds fully ionized and requires an enzyme residue (probably lysine) to be protonated. The maximum velocity and V/KMgATP are pH independent. The V/K for Ser-peptide is bell-shaped with pK values of 6.2 and 8.5 estimated. The pH dependence of 1/Ki for Leu-Arg-Arg-Ala-Ala-Leu-Gly is also bell-shaped, giving pK values identical with those obtained for V/KSer-peptide, while the Ki for MgAMP-PCP increases from a constant value of 650 microM above pH 8 to a constant value of 4 mM below pH 5.5. The Ki for uncomplexed Mg2+ obtained from the Mg2+ dependence of V and V/KMgATP is apparently pH independent.     

Chemical mechanism and rate-limiting steps in the reaction catalyzed by Streptococcus faecalis NADH peroxidase

The pH dependence of the kinetic parameters V, V/KNADH, and V/KH2O2 has been determined for the flavoenzyme NADH peroxidase. Both V/KNADH and V/KH2O2 decrease as groups exhibiting pK's of 9.2 and 9.9, respectively, are deprotonated. The V profile decreases by a factor of 5 as a group exhibiting a pK of 7.2 is deprotonated. Primary deuterium kinetic isotope effects on NADH oxidation are observed on V only, and the magnitude of DV is independent of H2O2 concentration at pH 7.5. DV/KNADH is pH independent and equal to 1.0 between pH 6 and pH 9.5, but DV is pH dependent, decreasing from a value of 7.2 at pH 5.5 to 1.9 at pH 9.5. The shape of the DV versus pH profile parallels that observed in the V profile and yields a similar pK of 6.6 for the group whose deprotonation decreases DV. Solvent kinetic isotope effects obtained with NADH or reduced nicotinamide hypoxanthine dinucleotide as the variable substrate are observed on V only, while equivalent solvent kinetic isotope effects on V and V/K are observed when H2O2 is used as the variable substrate. In all cases linear proton inventories are observed. Primary deuterium kinetic isotope effects on V for NADH oxidation decrease as the solvent isotopic composition is changed from H2O to D2O. These data are consistent with a change in the rate-limiting step from a step in the reductive half-reaction at low pH to a step in the oxidative half-reaction at high pH. Analysis of the multiple kinetic isotope effect data suggests that at high D2O concentrations the rate of a single proton transfer step in the oxidative half-reaction is slowed. These data are used to propose a chemical mechanism involving the pH-dependent protonation of a flavin hydroxide anion, following flavin peroxide bond cleavage.     

Inter-flavin electron transfer in cytochrome P450 reductase - effects of solvent and pH identify hidden complexity in mechanism

This study on human cytochrome P450 reductase (CPR) presents a comprehensive analysis of the thermodynamic and kinetic effects of pH and solvent on two- and four-electron reduction in this diflavin enzyme. pH-dependent redox potentiometry revealed that the thermodynamic equilibrium between various two-electron reduced enzyme species (FMNH*,FADH*; FMN,FADH2; FMNH2,FAD) is independent of pH. No shift from the blue, neutral di-semiquinone (FMNH*,FADH*) towards the red, anionic species is observed upon increasing the pH from 6.5 to 8.5. Spectrophotometric analysis of events following the mixing of oxidized CPR and NADPH (1 to 1) in a stopped-flow instrument demonstrates that the establishment of this thermodynamic equilibrium becomes a very slow process at elevated pH, indicative of a pH-gating mechanism. The final level of blue di-semiquinone formation is found to be pH independent. Stopped-flow experiments using excess NADPH over CPR provide evidence that both pH and solvent significantly influence the kinetic exposure of the blue di-semiquinone intermediate, yet the observed rate constants are essentially pH independent. Thus, the kinetic pH-gating mechanism under stoichiometric conditions is of no significant kinetic relevance for four-electron reduction, but rather modulates the observed semiquinone absorbance at 600 nm in a pH-dependent manner. The use of proton inventory experiments and primary kinetic isotope effects are described as kinetic tools to disentangle the intricate pH-dependent kinetic mechanism in CPR. Our analysis of the pH and isotope dependence in human CPR reveals previously hidden complexity in the mechanism of electron transfer in this complex flavoprotein.     

Kinetic and chemical mechanisms of the fabG-encoded Streptococcus pneumoniae beta-ketoacyl-ACP reductase

Beta-ketoacyl-acyl carrier protein reductase (KACPR) catalyzes the NADPH-dependent reduction of beta-ketoacyl-acyl carrier protein (AcAc-ACP) to generate (3S)-beta-hydroxyacyl-ACP during the chain-elongation reaction of bacterial fatty acid biosynthesis. We report the evaluation of the kinetic and chemical mechanisms of KACPR using acetoacetyl-CoA (AcAc-CoA) as a substrate. Initial velocity, product inhibition, and deuterium kinetic isotope effect studies were consistent with a random bi-bi rapid-equilibrium kinetic mechanism of KACPR with formation of an enzyme-NADP(+)-AcAc-CoA dead-end complex. Plots of log V/K(NADPH) and log V/K(AcAc)(-)(CoA) indicated the presence of a single basic group (pK = 5.0-5.8) and a single acidic group (pK = 8.0-8.8) involved in catalysis, while the plot of log V vs pH indicated that at high pH an unprotonated form of the ternary enzyme complex was able to undergo catalysis. Significant and identical primary deuterium kinetic isotope effects were observed for V (2.6 +/- 0.4), V/K(NADPH) (2.6 +/- 0.1), and V/K(AcAc)(-)(CoA) (2.6 +/- 0.1) at pH 7.6, but all three values attenuated to values of near unity (1.1 +/- 0.03 or 0.91 +/- 0.02) at pH 10. Similarly, the large alpha-secondary deuterium kinetic isotope effect of 1.15 +/- 0.02 observed for [4R-(2)H]NADPH on V/K(AcAc)(-)(CoA) at pH 7.6 was reduced to a value of unity (1.00 +/- 0.04) at high pH. The complete analysis of the pH profiles and the solvent, primary, secondary, and multiple deuterium isotope effects were most consistent with a chemical mechanism of KACPR that is stepwise, wherein the hydride-transfer step is followed by protonation of the enolate intermediate. Estimations of the intrinsic primary and secondary deuterium isotope effects ((D)k = 2.7, (alpha)(-D)k = 1.16) and the correspondingly negligible commitment factors suggest a nearly full expression of the intrinsic isotope effects on (D)V/K and (alpha)(-D)V/K, and are consistent with a late transition state for the hydride transfer step. Conversely, the estimated intrinsic solvent effect ((D)2(O)k) of 5.3 was poorly expressed in the experimentally derived parameters (D)2(O)V/K and (D)2(O)V (both = 1.2 +/- 0.1), in agreement with the estimation that the catalytic commitment factor for proton transfer to the enolate intermediate is large. Such detailed knowledge of the chemical mechanism of KAPCR may now help guide the rational design of, or inform screening assay-design strategies for, potent inhibitors of this and related enzymes of the short chain dehydrogenase enzyme class.     

Ionization of amino acid residues involved in the catalytic mechanism of aspartate transcarbamoylase.

The chemical and kinetic mechanisms of the reaction catalyzed by the catalytic trimer of aspartate transcarbamoylase have been examined. The variation of the kinetic parameters with pH indicated that at least four ionizing amino acid residues are involved in substrate binding and catalysis. The pH dependence of K(ia) for carbamoyl phosphate and the K(i) for N-(phosphonoacetyl)-L- aspartate revealed that a protonated residue with a pK value of 9.0 is required for the binding of carbamoyl phosphate. However, the variation with pH of K(i) for succinate, a competitive inhibitor of aspartate, and for cysteine sulfinate, a slow substrate, showed that a single residue with a pK value of 7.3 must be protonated for binding these analogues and, by inference, aspartate. The profile of log V against pH displayed a decrease in reaction rate at low and high pH, suggesting that two groups associated with the Michaelis complex, a deprotonated residue with a pK value of 7.2 and a protonated group with a pK value of 9.5, are involved in catalysis. By contrast, the catalytically productive form of the enzyme-carbamoyl phosphate complex, as illustrated in the bell-shaped pH dependence of log (V/K)(asp), is one in which a residue with a pK value of 7.0 must be protonated while a group with a pK value of 9.1 is deprotonated. This interpretation is supported by the results from the temperature dependence of the V and V/K profiles and from the pH dependence of pK(i) for the aspartate analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mycobacterium tuberculosis mycothione reductase: pH dependence of the kinetic parameters and kinetic isotope effects

The recent identification of the enzyme in Mycobacterium tuberculosis that catalyzes the NADPH-dependent reduction of the unique low molecular weight disulfide mycothione, mycothione reductase, has led us to examine the mechanism of catalysis in greater detail. The pH dependence of the kinetic parameters V and V/K for NADPH, NADH, and an active analogue of mycothione disulfide, des-myo-inositol mycothione disulfide, has been determined. An analysis of the pH profiles has allowed the tentative assignment of catalytically significant residues crucial to the mechanism of disulfide reduction, namely, the His444-Glu449 ion pair and Cys39. Solvent kinetic isotope effects were observed on V and V/K(DIMSSM), yielding values of 1.7 +/- 0.2 and 1.4 +/- 0.2, respectively, but not on V/K(NADPH). Proton inventory studies (V versus mole fraction of D(2)O) were linear, indicative of a single proton transfer in a solvent isotopically sensitive step. Steady-state primary deuterium kinetic isotope effects on V have been determined using NADPH and NADH, yielding values of 1.27 +/- 0.03 and 1.66 +/- 0.14, respectively. The pre-steady-state primary deuterium kinetic isotope effect on enzyme reduction has values of 1.82 +/- 0.04 and 1.59 +/- 0.06 for NADPH and NADH, respectively. The steady-state primary deuterium kinetic isotope effect using NADH coincide with that obtained under single turnover conditions, suggesting the complete expression of the intrinsic primary kinetic isotope effect. Rapid reaction studies on the reductive half-reaction using NADPH and NADH yielded maximal rates of 129 +/- 2 and 20 +/- 1 s(-1), respectively, while similar studies of the oxidation of the two-electron reduced enzyme by mycothiol disulfide yielded a maximum rate of 190 +/- 10 s(-1). These data suggest a unique flavoprotein disulfide mechanism in which the rate of the oxidative half-reaction is slightly faster than the rate of the reductive half-reaction.     

pH profiles and isotope effects for aconitases from Saccharomycopsis lipolytica, beef heart, and beef liver. alpha-Methyl-cis-aconitate and threo-Ds-alpha-methylisocitrate as substrates

alpha-Methyl-cis-aconitate (cis-2-butene-1,2,3-tricarboxylate) was converted only to alpha-methylisocitrate (3-hydroxybutane-1,2,3-tricarboxylate) by aconitases from beef liver or S. lipolytica. While the kinetic parameters of beef liver (cytoplasmic) or heart (mitochondrial) aconitases did not vary over the pH range 4.9-9 with the natural substrates, and only slightly with the alpha-methyl substrates, the yeast aconitase exhibited a bell-shaped pH profile with all substrates and for binding of the competitive inhibitor, tricarballylate, with pK values around 7 and 9. The third pK of the substrates does not affect V/K, showing that these pK's are for catalytic groups on the enzyme. One of these catalytic groups presumably removes a proton to give the carbanion intermediate in the reaction, and the other protonates the hydroxyl group when it is eliminated to give water, possibly with the assistance of the Fe-S center. Beef liver aconitase showed a primary deuterium isotope effect of 1.12 (measured by equilibrium perturbation with deuterated alpha-methylisocitrate) which was pH independent and only slightly greater than the equilibrium isotope effect. Isotope effects with the yeast enzyme were also pH independent but about 1.22 on V/K (or when measured by equilibrium perturbation) and 1.7 on V. These data suggest a kinetic mechanism for beef aconitases in which product release occurs only by displacement by the substrate in a step independent of pH or of the protonation state of the substrate. With the yeast enzyme, product displacement either depends on the protonation state of the catalytic groups on the enzyme or can occur spontaneously at a finite rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Ca(2+)-activated calmodulin on neuronal nitric oxide synthase reductase activity and binding of substrates: pH dependence of kinetic parameters.

The pH dependence of basal and calmodulin- (CaM-) stimulated neuronal nitric oxide synthase (nNOS) reduction of 2,6-dichloroindophenol (DCIP) and cytochrome c(3+) was investigated. The wave-shaped log V versus pH profile revealed that optimal DCIP reduction occurred when a group, pK(a) of 7.6-7.8, was ionized. The (V/K)(NADPH) and (V/K)(DCIP) versus pH profiles increased with the protonation of a group with a pK(a) of 6.5 or 5.9 and the ionization of two groups with the same pK(a) of 7.5 or 7.0, respectively. (V/K)(DCIP) decreased with the ionization of a group, pK(a) of 9.0. Similar V, (V/K)(NADPH), and (V/K)(DCIP) versus pH profiles for DCIP reduction were obtained with and without CaM, indicating that CaM does not influence ionizable groups involved in catalysis or substrate binding. In contrast, CaM affected the pH dependence of cytochrome c(3+) reduction. The wave-shaped log V versus pH profile for basal cytochrome c(3+) reduction revealed that ionization of a group, pK(a) of 8.6, increased catalysis. Log V for CaM-stimulated cytochrome c(3+) reduction displayed a bell-shaped pH dependence with the protonation of a group with a pK(a) of 6.4 and the ionization of a group with a pK(a) of 9.3, resulting in a loss of activity. The log(V/K)(cytc) versus pH profiles with and without CaM were bell-shaped with the ionization of a group at pK(a) of 7.1 or 7.6 (CaM) or pK(a) of 9.4 or 9.6 (CaM), increasing and decreasing (V/K)(cytc). These results suggest that CaM may change the nature of the rate-limiting catalytic steps or ionizable groups involved in cytochrome c(3+) reduction.     

A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae

Saccharopine dehydrogenase [N6-(glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine forming)] catalyzes the final step in the alpha-aminoadipate pathway for lysine biosynthesis. It catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate alpha-Kg and lysine using NAD+ as an oxidizing agent. The proton shuttle chemical mechanism is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. In the direction of lysine formation, once NAD+ and saccharopine bind, a group with a pKa of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized. This protonated general base then does not participate in the reaction again until lysine is formed at the completion of the reaction. A general base with a pKa of 7.2 accepts a proton from H2O as it attacks the Schiff base carbon of saccharopine to form the carbinolamine intermediate. The same residue then serves as a general acid and donates a proton to the carbinolamine nitrogen to give the protonated carbinolamine. Collapse of the carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate alpha-Kg and lysine. The amine nitrogen is then protonated by the group that originally accepted a proton from the secondary amine of saccharopine, and products are released. In the reverse reaction direction, finite primary deuterium kinetic isotope effects were observed for all parameters with the exception of V2/K(NADH), consistent with a steady-state random mechanism and indicative of a contribution from hydride transfer to rate limitation. The pH dependence, as determined from the primary isotope effect on DV2 and D(V2/K(Lys)), suggests that a step other than hydride transfer becomes rate-limiting as the pH is increased. This step is likely protonation/deprotonation of the carbinolamine nitrogen formed as an intermediate in imine hydrolysis. The observed solvent isotope effect indicates that proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple substrate/solvent isotope effects, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories are observed for V2 and V2/K(Lys), suggesting that proton transfer exists in at least two sequential transition states.     

Mechanistic studies of protein tyrosine phosphatases YopH and Cdc25A with m-nitrobenzyl phosphate

Protein tyrosine phosphatases (PTPs) constitute a large family of signaling enzymes that include both tyrosine specific and dual-specificity phosphatases that hydrolyze pSer/Thr in addition to pTyr. Previous mechanistic studies of PTPs have relied on the highly activated substrate p-nitrophenyl phosphate (pNPP), an aryl phosphate with a leaving group pK(a) of 7. In the study presented here, we employ m-nitrobenzyl phosphate (mNBP), an alkyl phosphate with a leaving group pK(a) of 14.9, which mimics the physiological substrates of the PTPs. We have carried out pH dependence and kinetic isotope effect measurements to characterize the mechanism of two important members of the PTP superfamily: Yersinia PTP (YopH) and Cdc25A. Both YopH and Cdc25A exhibit bell-shaped pH-rate profiles for the hydrolysis of mNBP, consistent with general acid catalysis. The slightly inverse (18)(V/K)(nonbridge) isotope effects (0.9999 for YopH and 0.9983 for Cdc25A) indicate a loose transition state with little nucleophilic participation for both enzymes. The smaller (18)(V/K)(bridge) primary isotope effects (0.9995 for YopH and 1.0012 for Cdc25A) relative to the corresponding isotope effects for pNPP hydrolysis suggest that protonation of the leaving group oxygen at the transition state by the general acid is ahead of P-O bond fission with the alkyl substrate, while general acid catalysis of pNPP by YopH is more synchronous with P-O bond fission. The isotope effect data also confirm findings from previous studies that Cdc25A utilizes general acid catalysis for substrates with a leaving group pK(a) of >8, but not for pNPP. Interestingly, the difference in the kinetic isotope effects for the reactions of aryl phosphate pNPP and alkyl phosphate mNBP by the PTPs parallels what is observed in the uncatalyzed reactions of their monoanions. In these reactions, the leaving group is protonated in the transition state, as is the case in PTP-catalyzed reactions. Also, the phosphoryl group in the transition states of the enzymatic reactions does not differ substantially from those of the uncatalyzed reactions. These results provide further evidence that these enzymes do not change the transition state but simply stabilize it.     

Kinetic and mechanistic analysis of the E. coli panE-encoded ketopantoate reductase

Ketopantoate reductase (EC 1.1.1.169) catalyzes the NADPH-dependent reduction of alpha-ketopantoate to form D-(-)-pantoate in the pantothenate/coenzyme A biosynthetic pathway. The enzyme encoded by the panE gene from E. coli K12 was overexpressed and purified to homogeneity. The native enzyme exists in solution as a monomer with a molecular mass of 34 000 Da. The steady-state initial velocity and product inhibition patterns are consistent with an ordered sequential kinetic mechanism in which NADPH binding is followed by ketopantoate binding, and pantoate release precedes NADP(+) release. The pH dependence of the kinetic parameters V and V/K for substrates in both the forward and reverse reactions suggests the involvement of a single general acid/base in the catalytic mechanism. An enzyme group exhibiting a pK value of 8.4 +/- 0.2 functions as a general acid in the direction of the ketopantoate reduction, while an enzyme group exhibiting a pK value of 7.8 +/- 0.2 serves as a general base in the direction of pantoate oxidation. The stereospecific transfer of the pro-S hydrogen atom of NADPH to the C-2 position of ketopantoate was demonstrated by (1)H NMR spectroscopy. Primary deuterium kinetic isotope effects of 1.3 and 1.5 on V(for) and V/K(NADPH), respectively, and 2.1 and 1.3 on V(rev) and V/K(HP), respectively, suggest that hydride transfer is not rate-limiting in catalysis. Solvent kinetic isotope effects of 1.3 on both V(for) and V/K(KP), and 1.4 and 1.5 on V(rev) and V/K(HP), respectively, support this conclusion. The apparent equilibrium constant, K(eq)', of 676 at pH 7.5 and the standard free energy change, DeltaG, of -14 kcal/mol suggest that ketopantoate reductase reaction is very favorable in the physiologically important direction of pantoate formation.     

Steady-state kinetics and isotope effects on the mutant catalytic trimer of aspartate transcarbamoylase containing the replacement of histidine 134 by alanine.

A detailed kinetic analysis of the catalytic trimer of aspartate transcarbamoylase containing the active site substitution H134A was performed to investigate the role of His 134 in the catalytic mechanism. Replacement of histidine by alanine resulted in decreases in the affinities for the two substrates, carbamoyl phosphate and aspartate, and the inhibitor succinate, by factors of 50, 10, and 6, respectively, and yielded a maximum velocity that was 5% that of the wild-type enzyme. However, the pK values determined from the pH dependence of the kinetic parameters, log V and log (V/K) for aspartate, the pK(i) for succinate, and the pK(ia) for carbamoyl phosphate, were similar for both the mutant and the wild-type enzymes, indicating that the protonated form of His 134 does not participate in binding and catalysis between pH 6.2 and 9.2. 13C and 15N isotope effects were studied to determine which steps in the catalytic mechanism were altered by the amino acid substitutions. The 13(V/K) for carbamoyl phosphate exhibited by the catalytic trimer containing alanine at position 134 revealed an isotope effect of 4.1%, probably equal to the intrinsic value and, together with quantitative analysis of the 15N isotope effects, showed that formation of the tetrahedral intermediate is rate-determining for the mutant enzyme. Thus, His 134 plays a role in the chemistry of the reaction in addition to substrate binding. The initial velocity pattern for the reaction catalyzed by the H134A mutant intersected to the left of the vertical axis, negating an equilibrium ordered kinetic mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dihydroorotate dehydrogenase from Clostridium oroticum is a class 1B enzyme and utilizes a concerted mechanism of catalysis

Dihydroorotate dehydrogenase from Clostridium oroticum was purified to apparent homogeneity and found to be a heterotetramer consisting of two alpha (32 kDa) and two beta (28 kDa) polypeptides. This subunit composition, coupled with known cofactor requirements and the ability to transfer electrons from L-dihydroorotate to NAD(+), defines the C. oroticum enzyme as a family 1B dihydroorotate dehydrogenase. The results of steady-state kinetic analyses and isotope exchange studies suggest that this enzyme utilizes a ping-pong steady-state kinetic mechanism. The pH-k(cat) profile is bell-shaped with a pK(a) of 6.4 +/- 0.1 for the ascending limb and 8. 9 +/- 0.1 for the descending limb; the pH-k(cat)/K(m) profile is similar but somewhat more complex. The pK(a) values of 6.4 and 8.9 are likely to represent the ionizations of cysteine and lysine residues in the active site which act as a general base and an electrostatic catalyst, respectively. At saturating levels of NAD(+), the isotope effects on (D)V and (D)(V/K(DHO)), obtained upon deuteration at both the C(5)-proR and C(5)-proS positions of L-dihydroorotate, increase from a value of unity at pH >9.0 to sizable values at low pH due to a high commitment to catalysis at high pH. At pH = 6.5, the magnitude of the double isotope effects (D)V and (D)(V/K(DHO)), obtained upon additional deuteration at C(6), is consistent with a mechanism in which C(5)-proS proton transfer and C(6)-hydride transfer occur in a single, partially rate-limiting step.     

Kinetics and mechanism of benzoylformate decarboxylase using 13C and solvent deuterium isotope effects on benzoylformate and benzoylformate analogues

Benzoylformate decarboxylase (benzoylformate carboxy-lyase, BFD; EC 4.1.1.7) from Pseudomonas putida is a thiamine pyrophosphate (TPP) dependent enzyme which converts benzoylformate to benzaldehyde and carbon dioxide. The kinetics and mechanism of the benzoylformate decarboxylase reaction were studied by solvent deuterium and 13C kinetic isotope effects with benzoylformate and a series of substituted benzoylformates (pCH3O, pCH3, pCl, and mF). The reaction was found to have two partially rate-determining steps: initial tetrahedral adduct formation (D2O sensitive) and decarboxylation (13C sensitive). Solvent deuterium and 13C isotope effects indicate that electron-withdrawing substituents (pCl and mF) reduce the rate dependence upon decarboxylation such that decreased 13(V/K) effects are observed. Conversely, electron-donating substituents increase the rate dependence upon decarboxylation such that a larger 13(V/K) is seen while the D2O effects on V and V/K are not dramatically different from those for benzoylformate. All of the data are consistent with substituent stabilization or destabilization of the carbanionic intermediate (or carbanion-like transition state) formed during decarboxylation. Additional information regarding the mechanism of the enzymic reaction was obtained from pH studies on the reaction of benzoylformate and the binding of competitive inhibitors. These studies suggest that two enzymic bases are required to be in the correct protonation state (one protonated and one unprotonated) for optimal binding of substrate (or inhibitors).     

Kinetic isotope effect analysis of the reaction catalyzed by Trypanosoma congolense trypanothione reductase.

African trypanosomes are devoid of glutathione reductase activity, and instead contain a unique flavoprotein variant, trypanothione reductase, which acts on a cyclic derivative of glutathione, trypanothione. The high degree of sequence similarity between trypanothione reductase and glutathione reductase, as well as the obvious similarity in the reactions catalyzed, led us to investigate the pH dependence of the kinetic parameters, and the isotopic behavior of trypanothione reductase. The pH dependence of the kinetic parameters V, V/K for NADH, and V/K for oxidized trypanothione has been determined for trypanothione reductase from Trypanosoma congolense. Both V/K for NADH and the maximum velocity decrease as single groups exhibiting pK values of 8.87 +/- 0.09 and 9.45 +/- 0.07, respectively, are deprotonated. V/K for oxidized trypanothione, T(S)2, decreases as two groups exhibiting experimentally indistinguishable pK values of 8.74 +/- 0.03 are deprotonated. Variable magnitudes of the primary deuterium kinetic isotope effects on pyridine nucleotide oxidation are observed on V and V/K when different pyridine nucleotide substrates are used, and the magnitude of DV and D(V/K) is independent of the oxidized trypanothione concentration at pH 7.25. Solvent kinetic isotope effects, obtained with 2',3'-cNADPH as the variable substrate, were observed on V only, and plots of V versus mole fraction of D2O (i.e., proton inventory) were linear, and yielded values of 1.3-1.6 for D2OV. Solvent kinetic isotope effects obtained with alternate pyridine nucleotides as substrates were also observed on V, and the magnitude of D2OV decreases for each pyridine nucleotide as its maximal velocity relative to that of NADPH oxidation decreases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of ionizable residues in the catalytic mechanism of tryptophan synthase from Salmonella typhimurium

The tryptophan synthase alpha2beta2 complex catalyzes the last two steps in the biosynthesis of l-tryptophan in bacteria, plants, and fungi, the conversion of indole-3-glycerol phosphate and l-serine to l-tryptophan, glyceraldehyde 3-phosphate, and water. The beta-subunit binds pyridoxal 5'-phosphate and catalyzes the beta-replacement reaction with serine and indole. Structural, spectral, and kinetic studies indicate that different monovalent cations stabilize the alternative enzyme conformations and equilibrium distribution of the internal, external, and alpha-aminoacrylate Schiff base. To improve our understanding of the role of monovalent cations, the pH dependence of steady-state and pre-steady-state kinetic parameters and primary kinetic deuterium isotope effects were measured in the presence of l-serine and [alpha-2H]-l-serine in the absence and presence of Na+, K+, and Cs+. For the interpretation of the data obtained in this study, it was necessary to re-interpret a number of results published previously. Overall, data suggest that the enzyme exists in two conformers that equilibrate slowly either in the absence of substrates and monovalent cations or in the presence of K+ or Cs+, whereas they equilibrate faster in the presence of Na+. The rate of interconversion of the conformers increases as a group on the enzyme with a pKa of approximately 8 becomes deprotonated. The pH dependence of deuterium isotope effects is suggestive of a mechanism in which a pH-dependent conformational change that closes the active site precedes the chemical steps, likely a result of formation of one or more salt bridges. As the pH increases, the reaction becomes more committed to proceed to products, which causes the deuterium isotope effect to decrease to a value of unity at high pH. The closure of the site is modulated by the different monovalent cations and is fastest in the presence of Na+, which exhibits the maximum isotope effect of 5.7 (likely the intrinsic effect) on V/Kserine, and slowest in the presence of Cs+, which exhibits the smallest isotope effect of approximately 1.5. The isotope effect on V, in all cases, indicates a contribution to rate limitation from steps in the second half of the reaction. Finally, in the presence of Na+, the steady-state isotope effect on V is greater than that on the pre-steady-state rate constant for decay of the external Schiff base, suggesting that the rate of conversion of the two conformers of the internal aldimine contributes to the pre-steady-state rate, but not the steady-state rate because the high serine concentration traps the enzyme in the active E-serine complex before it can decay to the less active form.