Peroxidase-catalyzed N-demethylation reactions. Substrate deuterium isotope effects

Deuterium isotope effects on the kinetic parameters for the hydroperoxide-supported N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase and horseradish peroxidase were determined using N,N-di-(trideuteromethyl)aniline. The isotope effect on the Vmax for the chloroperoxidase-catalyzed demethylation reaction supported by ethyl hydroperoxide was 1.42 +/- 0.31. The isotope effects on the Vmax for the horseradish peroxidase-catalyzed reaction supported by ethyl hydroperoxide and hydrogen peroxide were 1.99 +/- 0.39 and 4.09 +/- 0.27, respectively. Isotope effects ranging from 1.76 to 5.10 were observed on the Vmax/Km for the hydroperoxide substrate (i.e. the second order rate constant for the reaction of the hydroperoxide with the peroxidase to form compound I) in both enzyme systems when the N-methyl groups of N,N-dimethylaniline were deuterated. These results are not predicted by the simple ping-pong kinetic model for peroxidase-catalyzed N-demethylation reactions. The data are most simply explained by a mechanism involving the transfer of deuterium (or hydrogen) from N,N-dimethylaniline to the enzyme during catalysis. The deuterium must subsequently be displaced from the enzyme by the hydroperoxide, causing the observed isotope effects.     

Peroxidase-catalyzed N-demethylation reactions: deuterium solvent isotope effects

The effect of D2O on the kinetic parameters for the hydroperoxide-supported N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase and horseradish peroxidase was investigated in order to assess the roles of exchangeable hydrogens in the demethylation reaction. The initial rate of the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide exhibited a pL optimum (where L denotes H or D) of 4.5 in both H2O and D2O. The solvent isotope effect on the initial rate of the chloroperoxidase-catalyzed demethylation reaction was independent of pL, suggesting that the solvent isotope effect is not due to a change in the pK of a rate-controlling ionization in D2O. The solvent isotope effect on the Vmax for the chloroperoxidase-catalyzed demethylation reaction was 3.66 +/- 0.62. In contrast, the solvent isotope effect on the Vmax for the horseradish peroxidase catalyzed demethylation reaction was approximately 1.5 with either ethyl hydroperoxide or hydrogen peroxide as the oxidant, indicating that the exchange of hydrogens in the enzyme and hydroperoxide for deuterium in D2O has little effect on the rate of the demethylation reaction. The solvent isotope effect on the Vmax/KM for ethyl hydroperoxide in the chloroperoxidase-catalyzed demethylation reaction was 8.82 +/- 1.57, indicating that the rate of chloroperoxidase compound I formation is substantially decreased in D2O. This isotope effect is suggested to arise from deuterium exchange of the hydroperoxide hydrogen and of active-site residues involved in compound I formation. A solvent isotope effect of 2.96 +/- 0.57 was observed on the Vmax/KM for N,N-dimethylaniline in the chloroperoxidase-catalyzed reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isotopic probes of the argininosuccinate lyase reaction

The mechanism of the argininosuccinate lyase reaction has been probed by the measurement of the effects of isotopic substitution at the reaction centers. A primary deuterium isotope effect of 1.0 on both V and V/K is obtained with (2S,3R)-argininosuccinate-3-d, while a primary 15N isotope effect on V/K of 0.9964 +/- 0.0003 is observed. The 15N isotope effect on the equilibrium constant is 1.018 +/- 0.001. The proton that is abstracted from C-3 of argininosuccinate is unable to exchange with the solvent from the enzyme-intermediate complex but is rapidly exchanged with solvent from the enzyme-fumarate-arginine complex. A deuterium solvent isotope effect of 2.0 is observed on the Vmax of the forward reaction. These and other data have been interpreted to suggest that argininosuccinate lyase catalyzes the cleavage of argininosuccinate via a carbanion intermediate. The proton abstraction step is not rate limiting, but the inverse 15N primary isotope effect and the solvent deuterium isotope effect suggest that protonation of the guanidino group and carbon-nitrogen bond cleavage of argininosuccinate are kinetically significant.     

Mechanistic studies of the flavoenzyme tryptophan 2-monooxygenase: deuterium and 15N kinetic isotope effects on alanine oxidation by an L-amino acid oxidase

Tryptophan 2-monooxygenase (TMO) from Pseudomonas savastanoi catalyzes the oxidative decarboxylation of l-tryptophan during the biosynthesis of indoleacetic acid. Structurally and mechanistically, the enzyme is a member of the family of l-amino acid oxidases. Deuterium and 15N kinetic isotope effects were used to probe the chemical mechanism of l-alanine oxidation by TMO. The primary deuterium kinetic isotope effect was pH independent over the pH range 6.5-10, with an average value of 6.0 +/- 0.5, consistent with this being the intrinsic value. The deuterium isotope effect on the rate constant for flavin reduction by alanine was 6.3 +/- 0.9; no intermediate flavin species were observed during flavin reduction. The kcat/Kala value was 1.0145 +/- 0.0007 at pH 8. NMR analyses gave an equilibrium 15N isotope effect for deprotonation of the alanine amino group of 1.0233 +/- 0.0004, allowing calculation of the 15N isotope effect on the CH bond cleavage step of 0.9917 +/- 0.0006. The results are consistent with TMO oxidation of alanine occurring through a hydride transfer mechanism.     

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)     

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)     

Investigation of the mechanism of nicotine demethylation in Nicotiana through 2H and 15N heavy isotope effects: implication of cytochrome P450 oxidase and hydroxyl ion transfer

Heavy-atom isotope effects for the N-demethylation of nicotine have been determined in vivo in static-phase biosynthetically incompetent plant cell cultures of Nicotiana species. A (2)H kinetic isotope effect of 0.587 and a (15)N kinetic isotope effect of 1.0028 were obtained. An identical (15)N kinetic isotope effect of 1.0032 was obtained for the nicotine analogue, N-methyl-2-phenylpyrrolidine. The magnitude of the (15)N heavy-atom isotope effect indicates that the fission of the CN bond is not rate limiting for demethylation. The theoretical calculation of heavy-atom isotope effects for a model of the reaction pathway based on cytochrome P450 best fits the measured kinetic isotope effect to the addition of hydroxyl ion to iminium to form N-hydroxymethyl, for which the computed (2)H- and (15)N kinetic isotope effects are 0.689 and 1.0081, respectively. This large inverse (2)H kinetic isotope effect is not compatible with the initial abstraction of the H from the methyl group playing a significant kinetic role in the overall kinetic limitation of the reaction pathway, since computed values for this step (4.54 and 0.9995, respectively) are inconsistent with the experimental data.     

Use of multiple isotope effects to study the mechanism of 6-phosphogluconate dehydrogenase

The multiple isotope effect method of Hermes et al. [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106-5114] has been used to study the mechanism of the oxidative decarboxylation catalyzed by 6-phosphogluconate dehydrogenase from yeast. 13C kinetic isotope effects of 1.0096 and 1.0081 with unlabeled or 3-deuterated 6-phosphogluconate, plus a 13C equilibrium isotope effect of 0.996 and a deuterium isotope effect on V/K of 1.54, show that the chemical reaction after the substrates have bound is stepwise, with hydride transfer preceding decarboxylation. The kinetic mechanism of substrate addition is random at pH 8, since the deuterium isotope effect is the same when either NADP or 6-phosphogluconate or 6-phosphogluconate-3-d is varied at fixed saturating levels of the other substrate. Deuterium isotope effects on V and V/K decrease toward unity at high pH at the same time that V and V/K are decreasing, suggesting that proton removal from the 3-hydroxyl may precede dehydrogenation. Comparison of the tritium effect of 2.05 with the other measured isotope effects gives limits of 3-4 on the intrinsic deuterium and of 1.01-1.05 for the intrinsic 13C isotope effect for C-C bond breakage in the forward direction and suggests that reverse hydride transfer is 1-4 times faster than decarboxylation.     

Site of interaction between phenazine methosulphate and the respiratory chain of Bacillus subtilis.

The experimental data presented in this paper comprise kinetic deuterium isotope effects on acylation of papain with various substrates when conducted in H2O and 2H2O. With alkyl esters of N-acylamino acids there is no or very little isotope effect, whereas with N-acylamino acid amides the ratio kappa H2O/kappa 2H2O is less than 1, i.e. there is an inverse isotope effect. Similarly, alkylation of papain with methyl bromoacetate exhibits no kinetic isotope effect, whereas for the analogous alkylation with bromoacetamide an inverse isotope effect is observed. It is concluded that (a) general base catalysis does not occur in the acylation of papain and (b) kinetic deuterium isotope effects can be affected substantially by interaction between the substrate leaving group and the enzyme, which has not been considered in previous mechanistic investigations.     

Kinetic deuterium isotope effects for 7-alkoxycoumarin O-dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes. Rate-limiting C-H bond breaking in cytochrome P450 1A2 substrate oxidation

7-Ethoxy (OEt) coumarin has been used as a model substrate in many cytochrome P450 (P450) studies, including the use of kinetic isotope effects to probe facets of P450 kinetics. P450s 1A2 and 2E1 are known to be the major catalysts of 7-OEt coumarin O-deethylation in human liver microsomes. Human P450 1A2 also catalyzed 3-hydroxylation of 7-methoxy (OMe) coumarin at appreciable rates but P450 2E1 did not. Intramolecular kinetic isotope effects were used as estimates of the intrinsic kinetic deuterium isotope effects for both 7-OMe and 7-OEt coumarin dealkylation reactions. The apparent intrinsic isotope effect for P450 1A2 (9.4 for O-demethylation, 6.1 for O-deethylation) showed little attenuation in other competitive and noncompetitive experiments. With P450 2E1, the intrinsic isotope effect (9.6 for O-demethylation, 6.1 for O-deethylation) was attenuated in the noncompetitive intermolecular experiments. High noncompetitive intermolecular kinetic isotope effects were seen for 7-OEt coumarin O-deethylation in a baculovirus-based microsomal system and five samples of human liver microsomes (7.3-8.1 for O-deethylation), consistent with the view that P450 1A2 is the most efficient P450 catalyzing this reaction in human liver microsomes and indicating that the C-H bond-breaking step makes a major contribution to the rate of this P450 (1A2) reaction. Thus, the rate-limiting step appears to be the chemistry of the breaking of this bond by the activated iron-oxygen complex, as opposed to steps involved in the generation of the reactive complex. The conclusion about the rate-limiting step applies to all of the systems studied with this model P450 1A2 reaction including human liver microsomes, the most physiologically relevant.     

Evidence from nitrogen-15 and solvent deuterium isotope effects on the chemical mechanism of adenosine deaminase

We have determined 15N isotope effects and solvent deuterium isotope effects for adenosine deaminase using both adenosine and the slow alternate substrate 7,8-dihydro-8-oxoadenosine. With adenosine, 15N isotope effects were 1.0040 in H2O and 1.0023 in D2O, and the solvent deuterium isotope effect was 0.77. With 7,8-dihydro-8-oxoadenosine, 15N isotope effects were 1.015 in H2O and 1.0131 in D2O, and the solvent deuterium isotope effect was 0.45. The inverse solvent deuterium isotope effect shows that the fractionation factor of a proton, which is originally less than 0.6, increases to near unity during formation of the tetrahedral intermediate from which ammonia is released. Proton inventories for 1/V and 1/(V/K) vs percent D2O are linear, indicating that a single proton has its fractionation factor altered during the reaction. We conclude that a sulfhydryl group on the enzyme donates its proton to oxygen or nitrogen during this step. pH profiles with 7,8-dihydro-8-oxoadenosine suggest that the pK of this sulfhydryl group is 8.45. The inhibition of adenosine deaminase by cadmium also shows a pK of approximately 9 from the pKi profile. Quantitative analysis of the isotope effects suggests an intrinsic 15N isotope effect for the release of ammonia from the tetrahedral intermediate of approximately 1.03 for both substrates; however, the partition ratio of this intermediate for release of ammonia as opposed to back-reaction is 14 times greater for adenosine (1.4) than for 7,8-dihydro-8-oxoadenosine (0.1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Human erythrocyte glutathione reductase: chemical mechanism and structure of the transition state for hydride transfer

Kinetic parameters and primary deuterium kinetic isotope effects for NADH and five pyridine nucleotide substrates have been determined at pH 8.1 for human erythrocyte glutathione reductase. DV/KNADH and DV are equal to 1.4 and are pH independent below pH 8.1, but DV decreases to 1.0 at high pH as a group exhibiting a pK of 8.6 is deprotonated. This result suggests that as His-467' is deprotonated, the rate of the isotopically insensitive oxidative half-reaction is specifically decreased and becomes rate-limiting. For all substrates, equivalent V and V/K primary deuterium kinetic isotope effects are observed at pH values below 8.1. The primary deuterium kinetic isotope effect on V, but not V/K, is sensitive to solvent isotopic composition. The primary tritium kinetic isotope effects agree well with the corresponding value calculated from the primary deuterium kinetic isotope effects by using the Swain-Schaad relationship. This suggests that the primary deuterium kinetic isotope effects observed in these steady-state experiments are the intrinsic primary deuterium kinetic isotope effects for hydride transfer. The magnitude of the primary deuterium kinetic isotope effect is dependent on the redox potential of the pyridine nucleotide substrate used, varying from approximately 1.4 for NADH and -320 mV reductants to 2.7 for thioNADH to 4.2-4.8 for 3-acetylpyridine adenine dinucleotide (3APADH). The alpha-secondary tritium kinetic isotope effects also increase as the redox potential of the pyridine nucleotide substrate becomes more positive. Together, these data indicate that the transition state for hydride transfer is very early for NADH and becomes later for thioNADH and 3APADH, as predicted by Hammond's postulate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secondary isotope effects and structure-reactivity correlations in the dopamine beta-monooxygenase reaction: evidence for a chemical mechanism

The chemical mechanism of hydroxylation, catalyzed by dopamine beta-monooxygenase, has been explored with a combination of secondary kinetic isotope effects and structure-reactivity correlations. Measurement of primary and secondary isotope effects on Vmax/Km under conditions where the intrinsic primary hydrogen isotope effect is known allows calculation of the corresponding intrinsic secondary isotope effect. By this method we have obtained an alpha-deuterium isotope effect, Dk alpha = 1.19 +/- 0.06, with dopamine as substrate. The beta-deuterium isotope effect is indistinguishable from one. The large magnitude of Dk alpha, together with our previous determination of a near maximal primary deuterium isotope effect of 9.4-11, clearly indicates the occurrence of a stepwise process for C-H bond cleavage and C-O bond formation and hence the presence of a substrate-derived intermediate. To probe the nature of this intermediate, a structure-reactivity study was performed by using a series of para-substituted phenylethylamines. Deuterium isotope effects on Vmax and Vmax/Km parameters were determined for all of the substrates, allowing calculation of the rate constants for C-H bond cleavage and product dissociation and dissociation constants for amine and O2 loss from the enzyme-substrate ternary complex. Multiple regression analysis yielded an electronic effect of p = -1.5 for the C-H bond cleavage step, eliminating the possibility of a carbanion intermediate. A negative p value is consistent with formation of either a radical or a carbocation; however, a significantly better correlation is obtained with sigma p rather than sigma p+, implying formation of a radical intermediate via a polarized transition state. Additional effects determined from the regression analyses include steric effects on rate constants for substrate hydroxylation and product release and on KDamine, consistent with a sterically restricted binding site, and a positive electronic effect of p = 1.4 on product dissociation, ascribed to a loss of product from an enzyme-bound Cu(II)-alkoxide complex. These results lead us to propose a mechanism in which O-O homolysis [from a putative Cu(II)-OOH species] and C-H homolysis (from substrate) occur in a concerted fashion, circumventing the formation of a discrete, high energy oxygen species such as hydroxyl radical. The substrate and peroxide-derived radical intermediates thus formed undergo a recombination, kinetically limited by displacement of an intervening water molecule, to give the postulated Cu(II)-alkoxide product complex.     

Mechanism and activation for allosteric adenosine 5'-monophosphate nucleosidase. Kinetic alpha-deuterium isotope effects for the enzyme-catalyzed hydrolysis of adenosine 5'-monophosphate and nicotinamide mononucleotide

The kinetic alpha-deuterium isotope effect on Vmax/Km for hydrolysis of NMN catalyzed by AMP nucleosidase at saturating concentrations of the allosteric activator MgATP2- is kH/kD = 1.155 +/- 0.012. This value is close to that reported previously for the nonenzymatic hydrolysis of nucleosides of related structure, suggesting that the full intrinsic isotope effect for enzymatic NMN hydrolysis is expressed under these conditions; that is, bond-changing reactions are largely or completely rate-determining and the transition state has marked oxocarbonium ion character. The kinetic alpha-deuterium isotope effect for this reaction is unchanged when deuterium oxide replaces water as solvent, corroborating this conclusion. Furthermore, this isotope effect is independent of pH over the range 6.95-9.25, for which values of Vmax/Km change by a factor of 90, suggesting that the isotope-sensitive and pH-sensitive steps for AMP-nucleosidase-catalyzed NMN hydrolysis are the same. Values of kH/kD for AMP nucleosidase-catalyzed hydrolysis of NMN decrease with decreasing saturation of enzyme with MgATP2- and reach unity when the enzyme is less than half-saturated with this activator. This requires that the rate-determining step changes from cleavage of the covalent C-N bond to one which is isotope-independent. In contrast to the case for NMN hydrolysis, AMP nucleosidase-catalyzed hydrolysis of AMP at saturating concentrations of MgATP2- shows a kinetic alpha-deuterium isotope effect of unity. Thus, covalent bond-changing reactions are largely or completely rate-determining for hydrolysis of a poor substrate, NMN, but make little or no contribution to rate-determining step for hydrolysis of a good substrate, AMP, by maximally activated enzyme. This behavior has several precedents.     

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.     

The mechanism of glycogen synthetase as determined by deuterium isotope effects and positional isotope exchange experiments

The reaction mechanism for glycogen synthetase from rabbit muscle was examined by alpha-secondary deuterium isotope effects and positional exchange experiments. Incubation of glycogen synthetase with [beta-18O2,alpha beta-18O]UDP-Glc did not result in any detectable positional isotope exchange from the beta-nonbridge position to the anomeric oxygen of the glucose moiety. Glucono-1,5-lactone was found to be a noncompetitive inhibitor versus UDP-Glc. The kinetic constants, K(is) and K(ii), were found to be 91 +/- 4 microM and 0.70 +/- 0.09 mM, respectively. Deoxynojirimycin was a nonlinear inhibitor at pH 7.5. The alpha-secondary deuterium isotope effects were measured with [1-2H]UDP-Glc by the direct comparison method. The isotope effects on Vmax and Vmax/K were found to be 1.23 +/- 0.04 and 1.09 +/- 0.06, respectively. The inhibitory effects by glucono-lactone and deoxynojirimycon plus the large alpha-secondary isotope effect on Vmax have been interpreted to show that an oxocarbonium ion is an intermediate in this reaction mechanism. The lack of a detectable positional isotope exchange reaction in the absence of glycogen suggests the formation of a rigid tight ion pair between UDP and the oxocarbonium ion intermediate.     

Chemical mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae

Homoisocitrate dehydrogenase (HIcDH, 3-carboxy-2-hydroxyadipate dehydrogenase) catalyzes the fourth reaction of the alpha-aminoadipate pathway for lysine biosynthesis, the conversion of homoisocitrate to alpha-ketoadipate using NAD as an oxidizing agent. A chemical mechanism for HIcDH is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. According to the pH-rate profiles, two enzyme groups act as acid-base catalysts in the reaction. A group with a p K a of approximately 6.5-7 acts as a general base accepting a proton as the beta-hydroxy acid is oxidized to the beta-keto acid, and this residue participates in all three of the chemical steps, acting to shuttle a proton between the C2 hydroxyl and itself. The second group acts as a general acid with a p K a of 9.5 and likely catalyzes the tautomerization step by donating a proton to the enol to give the final product. The general acid is observed in only the V pH-rate profile with homoisocitrate as a substrate, but not with isocitrate as a substrate, because the oxidative decarboxylation portion of the isocitrate reaction is limiting overall. With isocitrate as the substrate, the observed primary deuterium and (13)C isotope effects indicate that hydride transfer and decarboxylation steps contribute to rate limitation, and that the decarboxylation step is the more rate-limiting of the two. The multiple-substrate deuterium/ (13)C isotope effects suggest a stepwise mechanism with hydride transfer preceding decarboxylation. With homoisocitrate as the substrate, no primary deuterium isotope effect was observed, and a small (13)C kinetic isotope effect (1.0057) indicates that the decarboxylation step contributes only slightly to rate limitation. Thus, the chemical steps do not contribute significantly to rate limitation with the native substrate. On the basis of data from solvent deuterium kinetic isotope effects, viscosity effects, and multiple-solvent deuterium/ (13)C kinetic isotope effects, the proton transfer step(s) is slow and likely reflects a conformational change prior to catalysis.     

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.     

Neuronal nitric oxide synthase: substrate and solvent kinetic isotope effects on the steady-state kinetic parameters for the reduction of 2,6-dichloroindophenol and cytochrome c(3+).

The neuronal nitric oxide synthase (nNOS) basal and calmodulin- (CaM-) stimulated reduction of 2,6-dichloroindophenol (DCIP) and cytochrome c(3+) follow ping-pong mechanisms [Wolthers and Schimerlik (2001) Biochemistry 40, 4722-4737]. Primary deuterium [NADPH(D)] and solvent deuterium isotope effects on the kinetic parameters were studied to determine rate-limiting step(s) in the kinetic mechanisms for the two substrates. nNOS was found to abstract the pro-R (A-side) hydrogen from NADPH. Values for (D)V and (D)(V/K)(NADPH) were similar for the basal (1.3-1.7) and CaM-stimulated (1.5-2.1) reduction of DCIP, while (D)V (2.1-2.8) was higher than (D)(V/K)(NADPH) (1.1-1.5) for cytochrome c(3+) reduction with and without CaM. This suggests that the rate of the reductive half-reaction (NADPH oxidation) rather than that of the oxidative half-reaction (reduction of DCIP or cytochrome c(3+)) limits the overall reaction rate. A value for (D)(V/K)(NADPH) close to 1 indicates the intrinsic isotope effect on hydride transfer is suppressed by a slower step in the reductive half-reaction. The oxidative half-reaction is insensitive to NADPD isotope effects as both (D)(V/K)(DCIP) and (D)(V/K)(cytc) equal 1 within experimental error. Large solvent kinetic isotope effects (SKIE) observed for (V/K)(cytc) for basal (approximately 8) and CaM-stimulated (approximately 31) reduction of cytochrome c(3+) suggest that proton uptake from the solvent limits the rate of the oxidative half-reaction. This step does not severely limit the overall reaction rate as (D2O)V equaled 2 and (D2O)(V/K)(NADPH) was between 0.9 and 1.3 for basal and CaM-stimulated cytochrome c(3+) reduction.