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.     

Human dihydrofolate reductase: reduction of alternative substrates, pH effects, and inhibition by deazafolates.

The kinetics of the NADPH-dependent reduction of 7,8-dihydrofolate, folate, and 7,8-dihydrobiopterin by human dihydrofolate reductase have been examined over the pH range from 4.0 to 9.5. The V and V/K profiles obtained with the three substrates indicate that a single ionizing residue at the active site of the enzyme must be protonated for catalysis. Both the maximum velocity of the reactions and the rate of interaction of the substrates with the enzyme-NADPH complex decrease in the order dihydrofolate greater than dihydrobiopterin much greater than folate. From the pK values of the V/K profiles, it can be concluded that, while dihydrofolate behaves as a sticky substrate and dihydrobiopterin exhibits slight stickiness, folate is not a sticky substrate. Further support for this conclusion comes from the results of deuterium isotope effects. The pK values obtained from both the V and V/Kfolate profiles are similar to the intrinsic pK value of 5.6 for both the free enzyme and the enzyme-NADPH complex. The folate analogue, 5-deazafolate, is not a substrate, but it undergoes strong interaction with the enzyme. This interaction, which is enhanced by the presence of NADPH, is due to protonation of the bound ligand that does not involve the single ionizing group at the active center of the enzyme. Difference spectra yield evidence for the protonation of bound 5-deazafolate and show that, on binding to the enzyme-NADPH complex, the pK of the N-8 atom is raised to about 10 from a value of about 4 in solution. The results are in accord with those of a recent paper on the three-dimensional structure of the enzyme-5-deazafolate complex [Davies, J.F., Delcamp, T.J., Prendergast, N.J., Ashfors, V.A., Freisheim, J.H., & Kraut, J. (1990) Biochemistry 29, 9467-9479] which indicate that there is hydrogen bond formation between N-8 of the ligand and the carbonyl group of Ile-7. However, the present findings do not support the idea that bound 5-deazafolate resembles the transition-state complex for folate reduction. Quinazolines also interact strongly with the enzyme but in a pH-independent manner. The dissociation constants for the binary complexes are an order of magnitude lower than that for the binding to the enzyme of unprotonated 5-deazafolate. This difference reflects the hydrophobic nature of the amino acid residues at the active site that are near the N-5 and N-8 nitrogens of bound pterins.     

Human liver aldehyde reductase: pH dependence of steady-state kinetic parameters.

The pH dependence of steady-state parameters for aldehyde reduction and alcohol oxidation were determined in the human liver aldehyde reductase reaction. The maximum velocity of aldehyde reduction with NADPH or 3-acetyl pyridine adenine dinucleotide phosphate (3-APADPH) was pH independent at low pH but decreased at high pH with a pK of 8.9-9.6. The V/K for both nucleotides decreased below a pK of 5.7-6.2, as did the pKi of competitive inhibitors NADP and ATP-ribose, suggesting that the 2'-phosphate of the nucleotide has to be deprotonated for binding to the enzyme. The pK of the 2'-phosphate of NADPH appears to be perturbed in the ternary complexes to 5.2-5.4. The V/K for NADPH, the V/K for 3-APADPH, and the pKi of ATP-ribose also decreased above a pK of 9-10, suggesting interaction of the 2'-phosphate of the nucleotide with a protonated base, perhaps lysine. Since protonation of a residue with a pK of 8 (evident in V/K for DL-glyceraldehyde and V/K for L-gulonate versus pH profiles) appears to be essential for aldehyde reduction, and deprotonation for alcohol oxidation, this residue appears to act as a general acid-base catalyst. An additional anion binding site with a pK of 9.94 facilitates the binding of carboxylic substrates such as D-glucuronate. With NADPH as the coenzyme the primary deuterium isotope effects on V and V/K for NADPH were close to unity and pH independent, suggesting that the hydride transfer step is not rate determining over the experimental pH range. With 3-APADPH as the coenzyme, the maximum velocity, relative to NADPH was three- to four-fold lower. Isotope effects on V, V/K for 3-APADPH, and V/K for D-glucuronate were pH independent and equal to 2.2-2.8, indicating that the chemical step of the reaction is relatively insensitive to pH. These data suggest that substrates bind to both the protonated and the deprotonated forms of the enzyme, though only the protonated enzyme catalyzes aldehyde reduction and the deprotonated enzyme catalyzes alcohol oxidation. On the basis of these results a scheme for the chemical mechanism of aldehyde reductase is postulated.     

Mechanism of modulation of dopamine beta-monooxygenase by pH and fumarate as deduced from initial rate and primary deuterium isotope effect studies

Dopamine beta-monooxygenase catalyzes a reaction in which 2 mol of protons are consumed for each turnover of substrate. Studies of the pH dependence of initial rate parameters (Vmax and Vmax/Km) and their primary hydrogen isotope effects show that at least two ionizable residues are involved in catalysis. One residue (B1, pK = 5.6-5.8) must be protonated prior to the carbon-hydrogen bond cleavage step, implying a role for general-acid catalysis in substrate activation. A second protonated residue (B2, pK = 5.2-5.4) facilitates, but is not required for, product release. Recent measurement of the intrinsic isotope effect for dopamine beta-monoxygenase [Miller, S. M., & Klinman, J. P. (1983) Biochemistry (preceding paper in this issue)] allows an analysis of the pH dependence of rate constant ratios and in selected instances individual rate constants. We demonstrate large changes in the rate-determining step as well as an unprecedented inversion in the kinetic order of substrate release from ternary complex over an interval of 2 pH units. Previously, fumarate has been used in dopamine beta-monooxygenase assays because of its property of enzyme activation. Studies of the pH behavior in the presence of saturating concentrations of fumarate have shown two causes of the activation: (i) fumarate perturbs the pK of B1 to pK = 6.6-6.8 such that the residue remains protonated and the enzyme optimally active over a wider pH range; (ii) fumarate decreases the rate of dopamine release from the ternary enzyme-substrate complex, increasing the equilibrium association constant for dopamine binding. Both effects are consistent with a simple electrostatic stabilization of bound cationic charges by the dianionic form of fumarate.     

Mechanism of nitroalkane oxidase: 2. pH and kinetic isotope effects

Nitroalkane oxidase catalyzes the oxidation of nitroalkanes to aldehydes or ketones with production of nitrite and hydrogen peroxide. pH and kinetic isotope effects with [1, 1-(2)H(2)]nitroethane have been used to study the mechanism of this enzyme. The V/K(ne) pH profile is bell-shaped. A group with a pK(a) value of about 7 must be unprotonated and one with a pK(a) value of 9.5 must be protonated for catalysis. The lower pK(a) value is seen also in the pK(is) profile for the competitive inhibitor valerate, indicating that nitroethane has no significant external commitments to catalysis. The (D)(V/K)(ne) value is pH-independent with a value of 7.5, whereas the (D)V(max) value increases from 1.4 at pH 8.2 to a limiting value of 7.4 below pH 5. The V(max) pH profile decreases at low and high pH, with pK(a) values of 6.6 and 9.5, respectively. Imidazole, which activates the enzyme, affects the V(max) but not the V/K(ne) pH profile. In the presence of imidazole at pH 7 the (D)V(max) value increases to a value close to the intrinsic value, consistent with cleavage of the carbon-hydrogen bond of the substrate being fully rate-limiting for catalysis in the presence of imidazole.     

Mechanistic deductions from kinetic isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Erwinia herbicola and Citrobacter freundii tyrosine phenol-lyase

The pH dependence of the kinetic parameters and primary deuterium isotope effects have been determined for tyrosine phenol-lyase from both Erwinia herbicola and Citrobacter freundii. The primary deuterium isotope effects indicate that proton abstraction from the 2-position of the substrate is partially rate-limiting for both enzymes. The C. freundii enzyme primary deuterium isotope effects [DV = 3.5 and D(V/Ktyr) = 2.5] are pH independent, indicating that tyrosine is not sticky (i.e., does not dissociate slower than it reacts to give products). Since Vmax for both tyrosine and the alternate substrate S-methyl-L-cysteine is also pH independent, substrate binds only to the correctly protonated form of the enzyme. For the E. herbicola enzyme, both Vmax and V/K for tyrosine or S-methyl-L-cysteine are pH dependent, as well as both DV and D(V/Ktyr). Thus, while both the protonated and unprotonated enzyme can bind substrate, and may be interconverted directly, only the unprotonated Michaelis complex is catalytically competent. At pH 9.5, DV = 2.5 and D(V/Ktyr) = 1.5. However, at pH 6.4 the isotope effect on both parameters is equal to 4.1. From these data, the forward commitment factor (cf = 5.2) and catalytic ratio (cvf = 1.1) for tyrosine and S-methyl-L-cysteine (cf = 2.2, cvf = 24) are calculated. Also, the Michaelis complex partition ratio (cf/cvf) for substrate and products is calculated to be 4.7 for tyrosine and 0.1 for S-methyl-L-cysteine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protonation mechanism and location of rate-determining steps for the Ascaris suum nicotinamide adenine dinucleotide-malic enzyme reaction from isotope effects and pH studies

The pH dependence of the kinetic parameters and the primary deuterium isotope effects with nicotinamide adenine dinucleotide (NAD) and also thionicotinamide adenine dinucleotide (thio-NAD) as the nucleotide substrates were determined in order to obtain information about the chemical mechanism and location of rate-determining steps for the Ascaris suum NAD-malic enzyme reaction. The maximum velocity with thio-NAD as the nucleotide is pH-independent from pH 4.2 to 9.6, while with NAD, V decreases below a pK of 4.8. V/K for both nucleotides decreases below a pK of 5.6 and above a pK of 8.9. Both the tartronate pKi and V/Kmalate decrease below a pK of 4.8 and above a pK of 8.9. Oxalate is competitive vs. malate above pH 7 and noncompetitive below pH 7 with NAD as the nucleotide. The oxalate Kis increases from a constant value above a pK of 4.9 to another constant value above a pK of 6.7. The oxalate Kii also increases above a pK of 4.9, and this inhibition is enhanced by NADH. In the presence of thio-NAD the inhibition by oxalate is competitive vs. malate below pH 7. For thio-NAD, both DV and D(V/K) are pH-independent and equal to 1.7. With NAD as the nucleotide, DV decreases to 1.0 below a pK of 4.9, while D(V/KNAD) and D(V/Kmalate) are pH-independent. Above pH 7 the isotope effects on V and the V/K values for NAD and malate are equal to 1.45, the pH-independent value of DV above pH 7. From the above data, the following conclusions can be made concerning the mechanism for this enzyme. Substrates bind to only the correctly protonated form of the enzyme. Two enzyme groups are necessary for binding of substrates and catalysis. Both NAD and malate are released from the Michaelis complex at equal rates which are equal to the rate of NADH release from E-NADH above pH 7. Below pH 7 NADH release becomes more rate-determining as the pH decreases until at pH 4.0 it completely limits the overall rate of the reaction.     

Kinetic isotope effects on the oxidation of reduced nicotinamide adenine dinucleotide phosphate by the flavoprotein methylenetetrahydrofolate reductase

Previous work from this laboratory has established that the NADPH-menadione oxidoreductase reaction catalyzed by methylenetetrahydrofolate reductase from pig liver proceeds by Ping Pong Bi Bi kinetics and that the reductive half-reaction is rate limiting in steady-state turnover. We have now shown that methylenetetrahydrofolate reductase stereo-specifically removes the pro-S hydrogen from the 4-position of NADPH. During the oxidation of [4(S)-3H]NADPH, we observed a kinetic isotope on V/KNADPH of 10.8 +/- 0.4. When comparing the rates of oxidation of [4(S)-2H]NADPH and [4(S)-1H]NADPH, we measure kinetic isotope effects on V of 4.78 +/- 0.15 and on V/KNADPH of 4.54 +/- 0.59. When oxidation of [4(R)-2H]NADPH and [4(R)-1H]NADPH is compared, the secondary kinetic isotope effect on V is 1.04 +/- 0.01. When the NADPH-menadione oxidoreductase reaction is catalyzed in tritiated water, no incorporation of solvent tritium into residual NADPH is observed. We conclude from these observations that the oxidation of NADPH is largely or entirely rate limiting in the reductive half-reaction and, hence, in NADPH-menadione oxidoreductase turnover at saturating menadione concentration. In the presence of saturating NADPH, the flavin reduction proceeds with a rate constant of 160 S-1, which is at least 29-fold slower than estimates of the lower limit for the diffusion-limited rate constant characterizing NADPH binding to the enzyme under physiological conditions. Albery & Knowles have defined criteria for perfection in enzyme catalysis [Albery, W. J., & Knowles, J.R. (1976) Biochemistry 15, 5631-5640].(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Glutathione reductase: comparison of steady-state and rapid reaction primary kinetic isotope effects exhibited by the yeast, spinach, and Escherichia coli enzymes

Kinetic parameters for NADPH and NADH have been determined at pH 8.1 for spinach, yeast, and E. coli glutathione reductases. NADPH exhibited low Km values for all enzymes (3-6 microM), while the Km values for NADH were 100 times higher (approximately 400 microM). Under our experimental conditions, the percentage of maximal velocities with NADH versus those measured with NADPH were 18.4, 3.7, and 0.13% for the spinach, yeast, and E. coli enzymes, respectively. Primary deuterium kinetic isotope effects were independent of GSSG concentration between Km and 15Km levels, supporting a ping-pong kinetic mechanism. For each of the three enzymes, NADPH yielded primary deuterium kinetic isotope effects on Vmax only, while NADH exhibited primary deuterium kinetic isotope effects on both V and V/K. The magnitude of DV/KNADH at pH 8.1 is 4.3 for the spinach enzyme, 2.7 for the yeast enzyme, and 1.6 for the E. coli glutathione reductase. The experimentally determined values of TV/KNADH of 7.4, 4.2, and 2.2 for the spinach, yeast, and E. coli glutathione reductases agree well with those calculated from the corresponding DV/KNADH using the Swain-Schaad expression. This suggests that the intrinsic primary kinetic isotope effect on NADH oxidation is fully expressed. In order to confirm this conclusion, single-turnover experiments have been performed. The measured primary deuterium kinetic isotope effects on the enzyme reduction half-reaction using NADH match those measured in the steady state for each of the three glutathione reductases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of methionine-13 in the catalytic mechanism of 6-phosphogluconate dehydrogenase from sheep liver

The crystal structure of sheep liver 6-phosphogluconate dehydrogenase (6PGDH) shows marked differences in the position of the nicotinamide mononucleotide (NMN) moiety of NADP(+) and NADPH (Adams, J. M., Grant, H. E., Gover, S., Naylor, C. E., and Phillips, C. (1994) Structure 2, 651-668). A methionine side chain (Met13) interacts with the si face of NADP(+) in the complex with the oxidized coenzyme, is likely to affect the binding mode of the nicotinamide ring of NADP(+), and may play a role in catalysis in the 6PGDH reaction. To check this possibility we performed site-directed mutagenesis, changing M13 to a number of residues including V, I, C, F, and Q. Mutant enzymes were characterized with respect to their kinetic parameters and primary deuterium isotope effects. All mutations resulted in a decrease in affinity of the enzyme for NADP(+), but not NADPH. In addition, the M13 to C (M13C), M13F, and M13Q mutant enzymes exhibited a decrease of at least an order of magnitude in V/E(t). The deuterium isotope effects on V and V/K(6PG) were decreased to about 1.2 for the M13F and M13C mutant enzymes, while they were increased to about 2.4 for the M13Q enzyme (a value of 1.8-1.9 is obtained for the wild-type enzyme). In at least three instances changes in the overall rate of the oxidative decarboxylation reaction relative to other steps along the reaction pathway were observed. Isotope effects indicate that the hydride transfer steps can become either more or less rate-determining dependent on the substitution. Data are consistent with a significant role of M13 in the orientation of the cofactor nicotinamide ring in the mechanism of 6PGDH, likely with respect to geometry and distance of the ring from C3 of 6PG.     

Solvent isotope effects on the reaction catalyzed by yeast hexokinase

The pH dependence of the maximum velocity (V) for the phosphorylation of glucose, the V/Kglucose and the V/KMgATP have been obtained in H2O and 2H2O. In H2O, V decreases below a pK of 5.8, V/Kglucose decreases below a pK of 6.1 and V/KMgATP decreases below a pK of 6.7. In 2H2O, complex behavior is observed for these parameters as a function of pD. The ratios of the parameters in H2O and 2H2O above their respective pK values give solvent deuterium isotope effects of about 1.5-1.7 for all three parameters. When 1,5-anhydromannitol is used as an alternative substrate, an isotope effect different than unity is obtained only for V/K1,5-anhydromannitol which gives a value of about 0.7. Both the complex pH profiles and the relative magnitude of the isotope effects are interpreted in terms of a pH-dependent change in the E X glucose complex.     

Mechanisms of enzymatic and acid-catalyzed decarboxylations of prephenate

The prephenate dehydrogenase activity of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase from Escherichia coli catalyzes the oxidative decarboxylation of both prephenate and deoxoprephenate, which lacks the keto group in the side chain (V 78% and V/K 18% those of prephenate). Hydride transfer is to the B side of NAD, and the acetylpyridine and pyridinecarboxaldehyde analogues of NAD have V/K values 40 and 9% and V values 107 and 13% those of NAD. Since the 13C isotope effect on the decarboxylation is 1.0103 with deuterated and 1.0033 with unlabeled deoxoprephenate (the deuterium isotope effect on V/K is 2.34), the mechanism is concerted, and if CO2 has no reverse commitment, the intrinsic 13C and deuterium isotope effects are 1.0155 (corresponding to a very early transition state for C-C bond cleavage) and 7.3, and the forward commitment is 3.7. With deoxodihydroprephenate (lacking one double bond in the ring), oxidation occurs without decarboxylation, and one enantiomer has a V/K value 23-fold higher than the other (deuterium isotope effects are 3.6 and 4.1 for fast and slow isomers; V for the fast isomer is 5% and V/K 0.7% those of prephenate). The fully saturated analogue of deoxoprephenate is a very slow substrate (V 0.07% and V/K approximately 10(-5%) those of prephenate). pH profiles show a group with pK = 8.3 that must be protonated for substrate binding and a catalytic group with pK = 6.5 that is a cationic acid (likely histidine). This group facilitates hydride transfer by beginning to accept the proton from the 4-hydroxyl group of prephenate prior to the beginning of C-C cleavage (or fully accepting it in the oxidation of the analogues with only one double bond or none in the ring). In contrast with the enzymatic reaction, the acid-catalyzed decarboxylation of prephenate and deoxoprephenate (t1/2 of 3.7 min at low pH) is a stepwise reaction with a carbonium ion intermediate, since 18O is incorporated into substrate and its epi isomer during reaction in H218O. pH profiles show that the hydroxyl group must be protonated and the carboxyl (pK approximately 4.2) ionized for carbonium ion formation. The carbonium ion formed from prephenate decarboxylates 1.75 times faster than it reacts with water (giving 1.8 times as much prephenate as epi isomer). The observed 13C isotope effect of 1.0082 thus corresponds to an intrinsic isotope effect of 1.023, indicating an early transition state for the decarboxylation step. epi-Prephenate is at least 20 times more stable to acid than prephenate because it exists largely as an internal hemiketal.(ABSTRACT TRUNCATED AT 400 WORDS)     

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)     

Use of nitrogen-15 and deuterium isotope effects to determine the chemical mechanism of phenylalanine ammonia-lyase

Phenylalanine ammonia-lyase has been shown to catalyze the elimination of ammonia from the slow alternate substrate 3-(1,4-cyclohexadienyl)alanine by an E1 cb mechanism with a carbanion intermediate. This conclusion resulted from comparison of 15N isotope effects with deuterated (0.9921) and unlabeled substrates (1.0047), and a deuterium isotope effect of 2.0 from dideuteration at C-3, with the equations for concerted, carbanion, and carbonium ion mechanisms. The 15N equilibrium isotope effect on the addition of the substrate to the dehydroalanine prosthetic group on the enzyme is 0.979, while the kinetic 15N isotope effect on the reverse of this step is 1.03-1.04 and the intrinsic deuterium isotope effect on proton removal is in the range 4-6. Isotope effects with phenylalanine itself are small (15N ones of 1.0021 and 1.0010 when unlabeled or 3-dideuterated and a deuterium isotope effect of 1.15) but are consistent with the same mechanism with drastically increased commitments, including a sizable external one (i.e., phenylalanine is sticky). pH profiles show that the amino group of the substrate must be unprotonated to react but that a group on the enzyme with a pK of 9 must be protonated, possibly to catalyze addition of the substrate to dehydroalanine. Incorrectly protonated enzyme-substrate complexes do not form. Equilibrium 15N isotope effects are 1.016 for the deprotonation of phenylalanine or its cyclohexadienyl analogue, 1.0192 for deprotonation of NH4+, 1.0163 for the conversion of the monoanion of phenylalanine to NH3, and 1.0138 for the conversion of the monoanion of aspartate to NH4+.(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.     

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 activation by malate induced by oxalate in the Ascaris suum NAD-malic enzyme reaction

Substrate activation of the rate of the NAD-malic enzyme reaction by malate is obtained in the presence but not in the absence of oxalate. The substrate activation is a result of competition between malate and oxalate for the E.NADH complex, with malate binding to the form of the complex unprotonated at an enzyme group with a pK of 4.9 and oxalate binding preferentially to the protonated form. The off-rate for NADH from the E.NADH complex is completely rate limiting when the group with a pK of 4.9 is protonated but is only one of several rate-limiting steps when it is unprotonated [Kiick, D.M., Harris, B.G., & Cook, P.F. (1986) Biochemistry 25, 227]. The competition by malate with oxalate thus results in an overall increase in the off-rate for NADH as a result of binding to the unprotonated form of E.NADH. Consistent with the proposed mechanism, the deuterium isotope effect on V for the nonsubstrate-activating malate concentration range decreases from 1.6 in the absence of oxalate to 1.3 in the presence of a concentration of oxalate equal to its Kii. The rate equation for the oxalate-induced substrate activation by malate is derived and presented in the Appendix. Data are discussed in terms of the overall mechanism of the NAD-malic enzyme.     

pH dependency of the reactions catalyzed by chorismate mutase-prephenate dehydrogenase from Escherichia coli

The variation with pH of the kinetic parameters associated with the mutase and dehydrogenase reactions catalyzed by chorismate mutase-prephenate dehydrogenase has been determined with the aim of elucidating the role that ionizing amino acid residues play in binding and catalysis. The pH dependency of log V for the dehydrogenase reaction shows that the enzyme possesses a single ionizing group with a pK value of 6.5 that must be unprotonated for catalysis. This same group is observed in the V/Kprephenate, as well as in the V/KNAD, profile. The V/Kprephenate profile exhibits a second ionizing residue with a pK value of 8.4 that must be protonated for the binding of prephenate to the enzyme. For the mutase reaction, the V/Kchorismate profile indicates the presence of three ionizing residues at the active site. Two of these residues, with similar pK values of about 7, must be protonated, while the third, with a pK value of 6.3, must be unprotonated. It can be concluded that all three groups are concerned with the binding of chorismate to the enzyme since the maximum velocity of the mutase reaction is essentially independent of pH. This conclusion is confirmed by the finding that the Ki profile for the competitive inhibitor, (3-endo,8-exo)-8-hydroxy-2-oxabicyclo[3.3]non-6-ene-3,5-dicarboxylic acid, shows the same three ionizing groups as observed in the V/Kchorismate profile. By contrast, the Ki profile for carboxyethyldihydrobenzoate shows only one residue, with a pK value of 7.3, that must be protonated for binding of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)