The necessity of magnesium cation for acid assistance aglycone departure in catalysis by Escherichia coli (lacZ) beta-galactosidase.

1. Removal of Mg2+ from Escherichia coli (lacZ) beta-galactosidase slightly increases the rate of hydrolysis of galactosyl pyridinium salts, but decreases the rate of hydrolysis of arylgalactosides. 2. Fair correlation of logkcat. and log (Km) with the pKa of aglycone is now observed for arglygalactosides, as well as for glycosyl pyridinium salts. 3. Degalactosylation of Mg2+-free enzyme is the rate-limiting step in the hydrolysis of 2,4-dinitrophenyl galactoside. 4. alpha-Deuterium kinetic isotope effects for both sets of substrates are consistent with the rate-determining generation of a glycosyl cation. 5. The pH-independent, SNl hydrolysis of 3,4-dinitrophenyl galactoside has been measured: it is as fast as that of the galactosyl 3-chloropyridinium ion. 6. Hydrolysis of these two substrates by Mg2+-free enzyme proceeds at very similar rates. 7. It is concluded that loss of both types of aglycone takes place, without acid catalysis, from the first ES complex of substrate and apoenzyme. 8. Data for galactosyl azide and thiopicrate confirm that neither charge nor change of atom is the cause of the differences in behavior between aryl galactosides and galactosylpyridinium salts.     

Mechanism of hydrolyses of phenyl alpha-maltosides catalyzed by taka-amylase A.

Michaelis constants (Kms) and molecular activities (kos) of phenyl, p-nitrophenyl and p-methylphenyl alpha-maltoside for taka-amylase A catalyzed hydrolyses were determined in H2O and in D2O at pH or pD 5.3 and at 25 degrees C. Production of alpha-maltose in the hydrolysis was confirmed by 1H NMR. Neither substituent nor solvent deuterium isotope effects on Kms for phenyl, p-nitrophenyl and p-methylphenyl alpha-maltosides were detected. On the other hand, substituent effects on kos of these compounds were evident, but the isotope effects on kos were not marked, so that protonation of the substrate in the catalytic reaction might not be rate-limiting. The result indicates that nucleophilic attack of a carboxylate anion of the enzyme upon the protonated substrate is the rate-limiting step in the hydrolysis proceeding through the nucleophilic double displacement mechanism, which involves a covalently bonded glycosyl intermediate. The molecular orbitals of phenyl alpha-D-glucosides as model compounds of phenyl alpha-maltosides were calculated by the AM1 method. From the results, it was concluded that the lowering of the lowest unoccupied molecular orbital (LUMO) energy levels and the increase of distribution of LUMO on the anomeric carbon, C-1, of the compounds are caused by protonation at the glycosidic oxygen from the protonated carboxyl group of the enzyme. This causes acceleration of the hydrolysis of a substrate by the enzyme.     

Hydrolysis of beta-galactosyl ester linkage by beta-galactosidases

p-Hydroxybenzoyl beta-galactose (pHB-Gal) was synthesized chemically to examine the hydrolytic activity of beta-galactosyl ester linkage by beta-galactosidases. The enzyme from Penicillium multicolor hydrolyzed the substrate as fast as p-nitrophenyl beta-galactoside (pNP-Gal), a usual substrate with a beta-galactosidic linkage. The enzymes from Escherichia coli and Aspergillus oryzae hydrolyzed pHB-Gal with almost the same rates as pNP-Gal. The enzymes from Bacillus circulans, Saccharomyces fragilis, and bovine liver showed much lower activities. pH-activity profiles, inhibition analysis, and kinetic properties of the enzymic reaction on pHB-Gal suggested that beta-galactosidase had only one active site for hydrolysis of both galactosyl ester and galactoside. The Penicillium enzyme hydrolyzed pHB-Gal in the presence of H218O to liberate galactose containing 18O. This result suggests the degradation occurs between the anomeric carbon and an adjacent O atom in the ester linkage of pHB-Gal.     

Mechanisms of acylation of chymotrypsin by phenyl esters of benzoic acid and acetic acid.

The kinetics of the acylation of alpha-chymotrypsin by a series of substituted phenyl p-nitrobenzoates have been studied by stopped flow and conventional spectrophotometry. Electron withdrawal in the leaving group accelerates the rate of acylation, and the p value obtained for eight esters is +1.96. The pH- and pD-independent acylation rate constants are, respectively, 1.40 X 10(4) M-1S-1 and 1.23 X 10(4) M-1S-1 for p-nitrophenyl p-nitrobenzoate, and, respectively, 2.19 X 10(3) M-1S-1 and 1968 X 10(3) M-1S-1 for p-nitrophenyl benzoate at 25 degrees. An analysis of structure-reactivity results and kinetic solvent isotope effects indicates a mechanism for acylation by phenylbenzoates in which initial reaction is a nucleophilic attack by an imidazole of the enzyme (His 57). Subsequently, there is rapid transfer of the acylating group to the serine 195 from the acylimidazole species. The kinetic solvent isotope effects for acylation by p-nitrophenyl phenyl acetate and p-nitrophenyl phenyl acetate and p-nitrophenyl hydrocinnamate, in 5%, v/v, acetonitrile, are 1.3 and 2.0, respectively. The latter ester is inhibited more than is p-nitrophenyl benzoate when 5%, v/v, dioxane is substituted for 5%, v/v, acetonitrile as co-solvent. In the presence of 5%, v/v, dioxane a change in the kinetic solvent isotope effect to 1.7 is found for p-nitrophenyl benzoate and p-nitrophenyl phenylacetate while that for the analogous hysdrocinnamate ester is unaffected. The results for the latter substrate are in accord with a general base-catalysed mechanism. Electron-withdrawal groups in the phenyl ring of phenyl acetates accelerate the enzyme acylation yielding a leaving group p of 2.05. The kinetic solvent isotope effects for acylation by p-nitrophenyl thiolacetate and by p-nitrophenyl acetate are close to 2.0. The mechanism of acylation of chymotrypsin by phenyl acetates is not unambiguously defined using these data.     

Comparison of the kinetic specificity of subtilisin and thiolsubtilisin toward n-alkyl p-nitrophenyl esters.

The p-nitrophenyl esters of straight-chain fatty acids were used as substrates of the enzyme subtilisin Novo (EC 3.4.4.16) and its chemically produced artificial enzyme thiolsubtilisin. Subtilisin and thiolsubtilisin pH--activity profiles were determined, and kinetic effects of the active site O-S substitution were observed. Among the substrates tested, both enzymes show highest specificity with p-nitrophenyl butyrate. It was also found that subtilisin is more sensitive to changes in substrate chain length than is thiolsubtilisin. Second-order acylation rate constants (k2/Ks) are remarkably similar for both enzymes. However, thiolsubtilisin deacylation rate constants and Km values are lower than analogous subtilisin constants. While thiolsubtilisin deacylation rate constants give a pH profile identical with that of subtilisin, the pH profile of thiolsubtilisin acylation rate constants shows an active site pK value lowered from the subtilisin pK of 7.15 and exhibits an inflection point at pH 8.45, which is absent in subtilisin.     

Transition-state structures for enzymatic and alkaline phosphotriester hydrolysis.

The primary and secondary 18O isotope effects for the alkaline (KOH) and enzymatic (phosphotriesterase) hydrolysis of two phosphotriesters, O,O-diethyl p-nitrophenyl phosphate (I) and O,O-diethyl O-(4-carbamoylphenyl) phosphate (II), are consistent with an associative mechanism with significant changes in bond order to both the phosphoryl and phenolic leaving group oxygens in the transition state. The synthesis of [15N, phosphoryl-18O]-, [15N, phenolic-18O]-, and [15N]-O,O-diethyl p-nitrophenyl phosphate and O,O-diethyl O-(4-carbamoylphenyl)phosphate is described. The primary and secondary 18O isotope effects for the alkaline hydrolysis of compound I are 1.0060 and 1.0063 +/- 0.0001, whereas for compound II they are 1.027 +/- 0.002 and 1.025 +/- 0.002, respectively. These isotope effects are consistent with the rate-limiting addition of hydroxide and provide evidence for a SN2-like transition state with the absence of a stable phosphorane intermediate. For the enzymatic hydrolysis of compound I, the primary and secondary 18O isotope effects are very small, 1.0020 and 1.0021 +/- 0.0004, respectively, and indicate that the chemical step in the enzymatic mechanism is not rate-limiting. The 18O isotope effects for the enzymatic hydrolysis of compound II are 1.036 +/- 0.001 and 1.0181 +/- 0.0007, respectively, and are comparable in magnitude to the isotope effects for alkaline hydrolysis, suggesting that the chemical step is rate-limiting. The relative magnitude of the primary 18O isotope effects for the alkaline and enzymatic hydrolysis of compound II reflect a transition state that is more progressed for the enzymatic reaction.     

The mechanism of the phosphoryl transfer catalyzed by Yersinia protein-tyrosine phosphatase: a computational and isotope effect study.

In order to evaluate various mechanistic proposals that have been made regarding the mechanism of the first step of the reaction catalyzed by protein-tyrosine phosphatases, new experimental data have been obtained, and some existing data have been carefully reevaluated. New kinetic isotope effect data for the uncatalyzed hydrolysis of p-nitrophenyl phosphate allow a better evaluation of previously reported data from enzymatic reactions with this substrate. The interpretation, and misinterpretation, of pH rate studies is considered. The pathway of this reaction has been modeled computationally and is found to be generally consistent with experimental studies, except for the extent of proton transfer to the leaving group.     

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.     

Solvent isotope effects on alpha-glucosidase

The solvent kinetic isotope effects (SKIE) on the yeast alpha-glucosidase-catalyzed hydrolysis of p-nitrophenyl and methyl-d-glucopyranoside were measured at 25 degrees C. With p-nitrophenyl-D-glucopyranoside (pNPG), the dependence of k(cat)/K(m) on pH (pD) revealed an unusually large (for glycohydrolases) solvent isotope effect on the pL-independent second-order rate constant, (DOD)(k(cat)/K(m)), of 1.9 (+/-0.3). The two pK(a)s characterizing the pH profile were increased in D(2)O. The shift in pK(a2) of 0.6 units is typical of acids of comparable acidity (pK(a)=6.5), but the increase in pK(a1) (=5.7) of 0.1 unit in going from H(2)O to D(2)O is unusually small. The initial velocities show substrate inhibition (K(is)/K(m) approximately 200) with a small solvent isotope effect on the inhibition constant [(DOD)K(is)=1.1 (+/-0.2)]. The solvent equilibrium isotope effects on the K(is) for the competitive inhibitors D-glucose and alpha-methyl D-glucoside are somewhat higher [(DOD)K(i)=1.5 (+/-0.1)]. Methyl glucoside is much less reactive than pNPG, with k(cat) 230 times lower and k(cat)/K(m) 5 x 10(4) times lower. The solvent isotope effect on k(cat) for this substrate [=1.11 (+/-0. 02)] is lower than that for pNPG [=1.67 (+/-0.07)], consistent with more extensive proton transfer in the transition state for the deglucosylation step than for the glucosylation step.     

Kinetic and chemical mechanism of malate synthase from Mycobacterium tuberculosis

Malate synthase catalyzes the Claisen-like condensation of acetyl-coenzyme A (AcCoA) and glyoxylate in the glyoxylate shunt of the citric acid cycle. The Mycobacterium tuberculosis malate synthase G gene, glcB, was cloned, and the N-terminal His(6)-tagged 80 kDa protein was expressed in soluble form and purified by metal affinity chromatography. A chromogenic 4,4'-dithiodipyridine assay did not yield linear kinetics, but the generation of an active site-directed mutant, C619S, gave an active enzyme and linear kinetics. The resulting mutant exhibited kinetics comparable to those of the wild type and was used for the full kinetic analysis. Initial velocity studies were intersecting, suggesting a sequential mechanism, which was confirmed by product and dead-end inhibition. The inhibition studies delineated the ordered binding of glyoxylate followed by AcCoA and the ordered release of CoA followed by malate. The pH dependencies of k(cat) and k(cat)/K(gly) are both bell-shaped, and catalysis depends on a general base (pK = 5.3) and a general acid (pK = 9.2). Primary kinetic isotope effects determined using [C(2)H(3)-methyl]acetyl-CoA suggested that proton removal and carbon-carbon bond formation were partially rate-limiting. Solvent kinetic isotope effects on k(cat) suggested the hydrolysis of the malyl-CoA intermediate was also partially rate-limiting. Multiple kinetic isotope effects, utilizing D(2)O and [C(2)H(3)-methyl]acetyl-CoA, confirmed a stepwise mechanism in which the step exhibiting primary kinetic isotope effects precedes the step exhibiting the solvent isotope effects. We combined the kinetic data and the pH dependence of the kinetic parameters with existing structural and mutagenesis data to propose a chemical mechanism for malate synthase from M. tuberculosis.     

Purification and mechanistic properties of an extracellular alpha-L-arabinofuranosidase from Monilinia fructigena.

1. The alpha-L-arabinofuranosidase isoenzyme designated AFIII [Laborda, Archer, Fielding & Byrde (1974) J. Gen. Microbiol. 81, 151-163] was purified by sequential isoelectric focusing, hydrophobic chromatography, gel filtration and chromatofocusing. 2. The enzyme is a monomer of Mr 40,000. 3. On inactivation of the enzyme with 3H-labelled 1-alpha-L-arabinofuranosylmethyl-3-p-nitrophenyltriazene, 0.64 mol of alpha-L-arabinofuranosylmethyl residues/mol of enzyme is estimated to become attached to protein. 5. Neither first-order nor second-order rate constants for hydrolyses of aryl alpha-L-arabinofuranosides are dependent upon leaving-group acidity [beta lg(V) = -0.16 +/- 0.11; Beta lg(V/K) = -0.11 +/- 0.07; n = 7; delta pKa = 4.5] 6. Bond-breaking is nonetheless rate-limiting, as is shown by a value of 18(V) of 1.030 +/- 0.007 for the hydrolysis of p-nitrophenyl arabinoside. 7. Proton-donation to the leaving group is thus far advanced at the rate-limiting transition state for this enzyme. 8. Four alpha-L-arabinofuranosyl pyridinium salts are substrates, and an approximate beta lg(V) value of -0.9 can be estimated. 9. The absolute rate enhancement with the 4-bromoisoquinolinium salt, 2.5 X 10(9), is comparable with that observed with pyranosidases. 10. Ring-opening mechanisms can therefore be dismissed, even though they are known in the acid-catalysed hydrolysis of arabinofuranosides.     

Nature of oxygen activation in glucose oxidase from Aspergillus niger: the importance of electrostatic stabilization in superoxide formation.

Glucose oxidase catalyzes the oxidation of glucose by molecular dioxygen, forming gluconolactone and hydrogen peroxide. A series of probes have been applied to investigate the activation of dioxygen in the oxidative half-reaction, including pH dependence, viscosity effects, 18O isotope effects, and solvent isotope effects on the kinetic parameter Vmax/Km(O2). The pH profile of Vmax/Km(O2) exhibits a pKa of 7.9 +/- 0.1, with the protonated enzyme form more reactive by 2 orders of magnitude. The effect of viscosogen on Vmax/Km(O2) reveals the surprising fact that the faster reaction at low pH (1.6 x 10(6) M-1 s-1) is actually less diffusion-controlled than the slow reaction at high pH (1.4 x 10(4) M-1 s-1); dioxygen reduction is almost fully diffusion-controlled at pH 9.8, while the extent of diffusion control decreases to 88% at pH 9.0 and 32% at pH 5.0, suggesting a transition of the first irreversible step from dioxygen binding at high pH to a later step at low pH. The puzzle is resolved by 18O isotope effects. 18(Vmax/Km) has been determined to be 1.028 +/- 0.002 at pH 5.0 and 1.027 +/- 0.001 at pH 9.0, indicating that a significant O-O bond order decrease accompanies the steps from dioxygen binding up to the first irreversible step at either pH. The results at high pH lead to an unequivocal mechanism; the rate-limiting step in Vmax/Km(O2) for the deprotonated enzyme is the first electron transfer from the reduced flavin to dioxygen, and this step accompanies binding of molecular dioxygen to the active site. In combination with the published structural data, a model is presented in which a protonated active site histidine at low pH accelerates the second-order rate constant for one electron transfer to dioxygen through electrostatic stabilization of the superoxide anion intermediate. Consistent with the proposed mechanisms for both high and low pH, solvent isotope effects indicate that proton transfer steps occur after the rate-limiting step(s). Kinetic simulations show that the model that is presented, although apparently in conflict with previous models for glucose oxidase, is in good agreement with previously published kinetic data for glucose oxidase. A role for electrostatic stabilization of the superoxide anion intermediate, as a general catalytic strategy in dioxygen-utilizing enzymes, is discussed.     

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 effect of pH, temperature, and D2O on the activity of porcine synovial collagenase and gelatinase

The pH dependence of Vmax and Vmax/Km for hydrolysis of Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond by porcine synovial collagenase and gelatinase was determined in the pH range 5-10. Both enzymes exhibited bell-shaped dependencies on pH for these two kinetic parameters, indicating that activity is dependent on at least two ionizable groups, one of which must be unprotonated and the other protonated. For collagenase, Vmax/Km data indicate that in the substrate-free enzyme, these groups have apparent pK values of 7.0 and 9.5, while the Vmax profile indicates similar pK values of 6.8 and 10.1 for the enzyme-substrate complex. The corresponding pH profiles of gelatinase were similar to those of collagenase, indicating the importance of groups with apparent pK values of 5.9 and 10.0 for the free enzyme and 5.9 and 11.1 for the enzyme-substrate complex. When these kinetic constants were determined in D2O using the peptide substrate, there was no significant effect on Vmax or Km for collagenase or Km for gelatinase. However, there was a deuterium isotope effect of approximately 1.5 on Vmax for gelatinase. These results indicate that a proton transfer step is not involved in the rate-limiting step for collagenase, but may be limiting with gelatinase. The Arrhenius activation energies for peptide bond hydrolysis of the synthetic peptide as well as the natural substrates were also determined for both enzymes. The activation energy (81 kcal) for hydrolysis of collagen by collagenase was nine times greater than that determined for the synthetic substrate (9.2 kcal). In contrast, the activation energy for hydrolysis of gelatin by gelatinase (26.3 kcal) was only 2.4 times greater than that for the synthetic substrate (11 kcal).     

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)     

Mechanistic consequences of mutation of the active site nucleophile Glu 358 in Agrobacterium beta-glucosidase.

The replacement of the active site nucleophile Glu 358 in Agrobacterium beta-glucosidase by Asn and Gln by site-directed mutagenesis results in essentially complete inactivation of the enzyme, while replacement by Asp generates a mutant with a rate constant for the first step, formation of the glycosylenzyme, some 2500 times lower than that of the native enzyme. This low activity is shown to be a true property of the mutant and not due to contaminating wild-type enzyme by active site titration studies and also through studies of its thermal denaturation and of the pH dependence of the reaction catalyzed. Binding of ground-state inhibitors is affected relatively little by the mutation, while binding of transition-state analogues is greatly impaired, consistent with a principal role for Glu 358 being in transition-state stabilization, not substrate binding. Determination of kinetic parameters for a series of aryl glucosides revealed that the glycosylation step is rate determining for all these substrates in contrast to the native enzyme, where a switch from rate-limiting glycosylation to rate-limiting deglycosylation was observed as substrate reactivity was increased. These results coupled with secondary deuterium kinetic isotope effects of kH/kD = 1.17 and 1.12 measured for the 2,4-dinitrophenyl and p-nitrophenyl glucosides point to a principal role of the nucleophile in stabilizing the cationic transition states and in formation of the covalent intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Newly Evolved Enzymes. III Evolution of the ebg Repressor during Selection for Enhanced Lactase Activity

The evolution of lactose utilization by lacZ deletion strains of E. coli occurs via mutations in the ebg genes. We show that one kind of mutation in the regulatory gene ebgR results in a repressor which retains the ability to repress synthesis of ebg enzymes, but which permits 4.5-fold more ebg enzyme synthesis during lactose induction than does the wild-type repressor. A comparison between the growth rate of various ebg+ strains on lactose and the amount of ebg enzyme synthesized by these strains shows that the rate of enzyme synthesis permitted by the wild-type repressor is insufficient for growth on lactose as a sole carbon source by a cell with the most active ebg lactase yet isolated. We conclude, therefore, that the evolution of lactose utilization requires both a structural and a regulatory mutation.     

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.     

Detailed comparative analysis of the catalytic mechanisms of beta-N-acetylglucosaminidases from families 3 and 20 of glycoside hydrolases

Beta-N-acetylglucosaminidases are commonly occurring enzymes involved in the degradation of polysaccharides and glycoconjugates containing N-acetylglucosamine residues. Such enzymes have been classified into glycoside hydrolase families 3 and 20 and are believed to follow distinct chemical mechanisms. Family 3 enzymes are thought to follow a standard retaining mechanism involving a covalent glycosyl enzyme intermediate while family 20 enzymes carry out a substrate-assisted mechanism involving the transient formation of an enzyme-sequestered oxazoline or oxazolinium ion intermediate. Detailed mechanistic analysis of representatives of these two families provides support for these mechanisms as well as detailed insights into transition state structure. Alpha-secondary deuterium kinetic isotope effects of kH/kD = 1.07 and 1.10 for Streptomyces plicatus beta-hexosaminidase (SpHex) and Vibrio furnisii beta-N-acetylglucosaminidase (ExoII) respectively indicate transition states with oxocarbenium ion character in each case. Br?nsted plots for hydrolysis of a series of aryl hexosaminides are quite different in the two cases. For SpHex a large degree of proton donation is suggested by the relatively low value of beta(lg) (-0.29) on kcat/Km, compared with a beta(lg) of -0.79 for ExoII. Most significantly the Taft plots derived from kinetic parameters for a series of p-nitrophenyl N-acyl glucosaminides bearing differing levels of fluorine substitution in the N-acyl group are completely different. A very strong dependence (slope = -1.29) is seen for SpHex, indicating direct nucleophilic participation by the acetamide, while essentially no dependence (0.07) is seen for ExoII, suggesting that the acetamide plays purely a binding role. Taken together these data provide unprecedented insight into enzymatic glycosyl transfer mechanisms wherein the structures of both the nucleophile and the leaving group are systematically varied.     

Structure-function correlation of fatty acyl-CoA dehydrogenase and fatty acyl-CoA oxidase

We have employed a new pseudosubstrate, beta-(2-furyl)propionyl coenzyme A (FPCoA), to study the functional properties of two enzymes, fatty acyl-CoA dehydrogenase from porcine liver and fatty acyl-CoA oxidase from Candida tropicalis, involved in the oxidation of fatty acids. Previous studies from our laboratory have shown that the dehydrogenase exhibits oxidase activity at the rate of dissociation of the product charge-transfer complex. This raises the question of the difference in functionality between these two flavoproteins. To investigate these differences, we have compared the pH dependence of product formation, the isotope effects using tetradeuterio-FPCoA, and the spectral properties and chemical reactivity of the product charge-transfer complexes formed with the two enzymes. The pH dependencies of the reaction of FPCoA with electron-transfer flavoprotein (ETF) for the dehydrogenase and of the reaction of FPCoA with O2 for the oxidase are quite similar. Both reactions proceed more rapidly at basic pH values while substrate binds more tightly at acidic pH values. These data for both enzymes are consistent with a mechanism in which enzyme is involved in protonation of the carbonyl group of substrate followed by base-catalyzed removal of the C-2 proton from substrate. The C-2 anion of substrate may then serve as the active species in reduction of enzyme-bound flavin. The deuterium isotope effects for both enzyme systems are primary across the entire pH range, assuring that the chemically important step of substrate oxidation is rate limiting in these steady-state kinetic experiments. The two enzymes differ in the chemical reactivity of their product charge-transfer complexes.(ABSTRACT TRUNCATED AT 250 WORDS)