Solvent Deuterium Research Articles

The super-electrophilic reactivity of 4-nitro-6-trifluoromethanesulfonyl-benzofuroxan in aqueous solution

A kinetic and thermodynamic study of the covalent hydration of 4-nitro-6trifluoromethanesulfonylbenzofuroxan, 4, to give the corresponding hydroxy σ-adduct, C-4, in aqueous solution is reported. Analysis of the data obtained in the pH range 0.8–13 has allowed dissection of the observed rates into forward ( k OH 2 ) and reverse ( k , k , k O H 1 2 − + − H 1 -2) rate constants as well as the obtention of pKa values for water addition to the carbocyclic ring. The results reveal that C-4 is the most stable hydroxy σ-adduct known to date (pKa = 2.95) and that its formation arises exclusively from the attack of water molecules between pH 2.5 and 8.5. The related rate constant k is equal to (0.15±0.01) s O H 1 2 , as compared with k = 0.035 s O H 1 2 -1 for the hydration of 4,6-dinitrobenzofuroxan (DNBF, pKa = 3.75). These figures show that substitution of the 6-NO2 group of this latter compound by an SO2CF3 group appreciably increases the electrophilic character of the carbocyclic ring of the benzofuroxan structure. Another manifestation of the activating effect of the SO2CF3 group is that the OH group covalently bonded to the 7-carbon of C-4 undergoes ionization in dilute NaOH solutions (pKa’ = 12.03). Some data pertaining to buffer catalysis and solvent deuterium isotope effects are also reported. From these results, it is concluded that the formation and decomposition of C-4 proceeds through the same mechanisms as those identified in the hydration of DNBF, in particular with OH acting as a general base catalyst and CO acting as a nucleophilic catalyst. − 2 3

Analyses of kinetic solvent isotope effects of a hammerhead ribozyme reaction in NH4+ and Li+ ions.

Hammerhead ribozymes have been considered to be divalent-metalloenzymes. However, this was recently questioned by the finding that the reaction can proceed without any divalent metal ions in the presence of high concentrations of monovalent ions such as NH4+/Li+ ions. Thus, one might think divalent metal ions are not involved in chemical step in the catalytic mechanism. To investigate the involvement of the monovalent ions, we analyzed the deuterium solvent isotope effects in the reactions. Our present analysis indicates a proton transfer(s) occurs only in the NH(4+)-mediated reaction, not in the Li(+)-mediated. Most simple interpretation is that NH4+ works as a general acid catalyst and Li+ as Lewis acid catalyst. This suggests hammerhead ribozymes can change catalyst upon their surrounding conditions.

Aspects of the hydrolysis of formamide: revisitation of the water reaction and determination of the solvent deuterium kinetic isotope effect in base

A study of the hydrolysis of formamide is reported with the aims of isolating the water reaction for hydrolysis from the acid and base hydrolysis terms and determining the solvent deuterium kinetic isotope effect (dkie) on base-catalyzed hydrolysis. Respective activation parameters (ΔH‡and ΔS‡) of (17.0 ± 0.4) kcal mol–1and (–18.8 ± 1.3) cal mol–1K–1for the acid reaction and (17.9 ± 0.2) kcal mol–1and (–11.1 ± 0.5) cal mol–1K–1for the base reaction were determined from Eyring plots of the second-order rate constants over the range of 27–120°C. Kinetic studies at the minima of the pH/rate profiles in the pH range from 5.6 to 6.2 in MES buffers at 56°C, and in the pH range of 4.25–6.87 in acetate and phosphate buffers at 120°C are reported. At 56°C the available data fit the expression k56obs= 0.00303[H3O+] + 0.032[HO–] + (3.6 ± 0.1) × 10–9, while at 120°C the data fit k120obs= (0.15 ± 0.02)[H3O+] + (3.20 ± 0.24)[HO–] + (1.09 ± 0.29) × 10–6. Preliminary experimental estimates of Ea(ln A) of 22.5 kcal mol–1(15.03) for the water rate constant (kw) are calculated from an Arrhenius plot of the 56 and 120°C data giving an estimated kwof 1.1 × 10–10s–1(t1/2= 199 years) at 25°C. Solvent dkie values of kOH/kOD= 1.15 and 0.77 ± 0.06 were determined at [OL–] = 0.075 and 1.47 M, respectively. The inverse value is determined under conditions where the the first step of the reaction dominates and is analyzed in terms of a rate-limiting attack of OL–.Key words: formamide, activation parameters, water reaction, acid and base hydrolysis, solvent kinetic isotope effect.

Tuning the redox chemistry of 4-benzoyl-N-methylpyridinium cations through para substitution. Hammett linear free energy relationships and the relative aptitude of the two-electron reduced forms for H-bonding.

In anhydrous CH3CN a series of nine 4-(4-substituted-benzoyl)-N-methylpyridinium cations (substituent: -OCH3, -CH3, -H, -SCH3, -Br, -Ctbd1;CH, -CHO, -NO2, and -(+)S(CH3)2) demonstrate two chemically reversible, well-separated one-electron (1-e) reductions in the same potential range as other main stream redox catalysts such as quinones and viologens. Hammett linear free energy plots yield excellent correlation between the E(1/2) values of both waves and the substituent constants sigma(p)(-)(X). The reaction constants for the two 1-e reductions are rho(1) = 2.60 and rho(2) = 3.31. The lower rho(1) value is associated with neutralization of the pyridinium ring, and the higher rho(2) value with the negative charge developing during the 2nd-e reduction. Structure-function correlations point to a purely inductive role for substitution in both 1-e reductions. The case of the 4-(4-nitrobenzoyl)-N-methylpyridinium cation is particularly noteworthy, because the 4-nitrobenzoyl moiety undergoes reduction before the 2nd reduction of the 4-benzoyl-N-methylpyridinium system. Correlation of the third wave of this compound with the 2nd-e reduction of the others yields sigma(p)(-NO)2*- = -0.97 +/- 0.02, thus placing the -NO2-* group among the strongest electron donors. Solvent deuterium isotope effects and maps of the electrostatic potential (via PM3 calculations) as a function of substitution support that 2-e reduced forms develop H-bonding with proton donors (e.g., CH3OH) via the O-atom. The average number of CH3OH molecules entering the H-bonding association increases with e-donating substituents. H-bonding shifts the 2nd reduction wave closer to the first one. This has important practical implications, because it increases the equilibrium concentration of the 2-e reduced form from disproportionation of the 1-e reduced form.

Evidence for direct attack by hydroxide in phosphodiester hydrolysis.

Phosphodiester hydrolysis has been the subject of intense study due to its importance in biology. Despite the numerous significant analyses of phosphodiester cleavage mechansim, comparatively little is known about the nucleophiles in these reactions. To determine whether hydroxide acts as a nucleophile or a general base in the hydrolysis of thymidine-5'-p-nitrophenyl phosphate,we determined solvent deuterium isotope effects (D2Ok), ionic strength effects, and 18O isotope effects on the solvent nucleophile (18knuc). The D2Ok for hydroxide-catalyzed phosphodiester hydrolysis is slightly inverse (0.9 +/- 0.1), suggesting that a proton transfer does not occur in the transition state. A significant alpha effect is observed with hydroperoxide, demonstrating that oxyanions can act as nucleophiles in the reaction. Additionally, the ionic strength dependencies of hydroxide and hydroperoxide catalysis are indistinguishable, suggesting that they act by the same mechanism. Finally, the 18knuc for the hydroxide-catalyzed reaction is 1.068 +/- 0.007, well in excess of the equilibrium 18O isotope effect between water and hydroxide (1.040 +/- 0.003). Together, the data are most consistent with direct nucleophilic attack by hydroxide. From the observed 18knuc and the known equilibrium component, the kinetic component of the isotope effect was calculated to be 1.027 +/- 0.010. This large kinetic component suggests that little bond order to the nucleophile occurs in the transition state.

Acid-Catalysed Hydrolysis of Some Aromatic Cyclic Sulfamates

The acid catalysed hydrolysis of some cyclic sulfamates, X-3-(p-tolylsulfonyl)-1,2,3-benzoxathiazole 2,2-dioxides ( 1a , X = Me; 1b , X = H; 1c , X = Cl; 1d , X = NO 2 ) have been studied in concentrated aqueous sulfuric and perchloric acid solutions. Analysis of the data by the Excess Acidity Method, activation parameters, substituent, solvent deuterium isotope effect and order of the catalytic effects of the acids are all in agreement with an A-1 mechanism in the studied range.

Water and hydroxide ion pathways in the σ-complexation of superelectrophilic 2-aryl-4,6-dinitrobenzotriazole 1-oxides in aqueous solution. A kinetic and thermodynamic study

As part of our continuing studies of the highly electron-deficient nature of nitrobenzofuroxans, nitrobenzofurazans and related heterocycles, we report here a kinetic and thermodynamic study of σ-complexation for a series of 2-aryl-4,6 -dinitrobenzotriazole 1-oxides (3a–e) over a large pH range in aqueous solution. The reaction series represents a modulation in electrophilic properties of the benzotriazole moiety in formation of the corresponding hydroxy σ-adducts (4a–e). Analysis of the data has allowed dissection of observed rates into forward (kH2O1, kOH−2) and reverse (kH+−1, k−2) rate constants as well as the obtention of pKa values for H2O addition to the benzotriazole moiety. Our results reveal that 3a–e are superelectrophilic compounds with respect to 1,3,5-trinitrobenzene (TNB) as a standard electron-deficient aromatic, but less superelectrophilic compared to 4,6-dinitrobenzofuroxan (DNBF). Some data pertaining to buffer catalysis of the formation and decomposition of the adducts together with solvent deuterium isotope effects for these pathways are also reported. From these results, it is concluded that adduct formation occurs via general base catalyzed water attack: the general bases include notably H2O, HCO3−, CO32− as well as OH−. This contrasts with the situation for the σ-complexation of DNBF where HCO3− and CO32− were found to act as nucleophilic catalysts whereas OH− functioned as a general base catalyst. This contrasting behaviour provides further evidence that the dinitro-activated carbocyclic ring of the benzotriazoles 3a–e ranks somewhat lower in electrophilic/superelectrophilic properties compared to that in DNBF. Altogether, the results provide a basis for understanding the relationship between the superelectrophilic reactivities, as evidenced by the contrasting kinetic and thermodynamic properties of the systems at hand, and the varied abilities of these substrates to react in pericyclic Diels–Alder reactions.

Chain and monomolecular decomposition of hexogen in solutions

The decomposition of hexogen dissolved in alkylaromatic hydrocarbons, alcohols, ketones, ethers, chloroform, and some other solvents occurs via the chain mechanism. This mechanism is supported by slowing down of the reaction when inhibitors are added, the solvent deuterium kinetic isotope effect, and the dependence of the rate on the reactivity of the C—H bond in solvents. The chain reaction propagates through the transfer of a free valence from the primary N-radicals formed by N—NO2 bond dissociation to the C-centered radicals of the solvent. The solvents are inert when the N—H bond dissociation energy is >380 or <200 kJ mol–1, and hexogen decomposition in such solvents is monomolecular.

Acid-catalyzed hydrolysis of bridged bi- and tricyclic compounds. XXXIX?Kinetics and mechanisms of the hydration reactions of 1- and 3-acetylnortricyclanes

The disappearance of 1- and 3-acetylnortricyclanes (1-Ac and 2-Ac) in aqueous perchloric acid was followed by capillary gas chromatography at different temperatures and acid concentrations. 1-Ac is much less reactive than 2-Ac. The activation parameters, solvent deuterium isotope effects and parameters of excess acidity equations were measured and the products studied. 1-Acetylnortricyclane is hydrated according to the A-2 mechanism, i.e. the carbonyl oxygen is protonated in the fast pre-equilibrium and one water molecule attacks at the rate-limiting stage the partially open cyclopropane ring, producing 6-acetyl-2-norborneols. 3-Acetylnortricyclane is hydrated according to the AdE2 mechanism, i.e. the cyclopropane ring is slowly protonated and opened, with subsequent fast attack of water producing 3-, 5- and 7-acetyl-2-norborneols. Copyright © 2002 John Wiley & Sons, Ltd.

Enzymatic hydrolysis of p-nitroacetanilide: mechanistic studies of the aryl acylamidase from Pseudomonas fluorescens.

Aryl acylamidase (EC 3.1.5.13; AAA) catalyzes the hydrolysis of p-nitroacetanilide (PNAA) via the standard three-step mechanism of serine hydrolases: binding of substrate (K(s)), acylation of active-site serine (k(acyl)), and hydrolytic deacylation (k(deacyl)). Key mechanistic findings that emerged from this study include that (1) AAA requires a deprotonated base with a pK(a) of 8.3 for expression of full activity toward PNAA. Limiting values of kinetic parameters at high pH are k(c) = 7 s(-1), K(m) = 20 microM, and k(c)/K(m) = 340 000 M(-1) s(-1). (2) At pH 10, where all the isotope effects were conducted, k(c) is equally rate-limited by k(acyl) and k(deacyl). (3) The following isotope effects were determined: (D)()2(O)(k(c)/K(m)) = 1.7 +/- 0.2, (D)()2(O)k(c) = 3.5 +/- 0.3, and (beta)(D)(k(c)/K(m)) = 0.83 +/- 0.04, (beta)(D)k(c) = 0.96 +/- 0.01. These values, together with proton inventories for k(c)/K(m) and k(c), suggest the following mechanism: (i) The initial binding of substrate to enzyme to form the Michaelis complex is accompanied by solvation changes that generate solvent deuterium isotope effects originating from hydrogen ion fractionation at multiple sites on the enzyme surface. (ii) From within the Michaelis complex, the active site serine attacks the carbonyl carbon of PNAA with general-base catalysis to form a substantially tetrahedral transition state enroute to the acyl-enzyme. (iii) Finally, deacylation occurs through a process involving a rate-limiting solvent isotope effect, generating conformational change of the acyl-enzyme that positions the carbonyl bond in a polarizing environment that is optimal for attack by water.

Solvent kinetic isotope effects monitor changes in hydrogen bonding at the active center of yeast pyruvate decarboxylase concomitant with substrate activation: the substituent at position 221 can control the state of activation.

Substrate activation of yeast pyruvate decarboxylase has been studied extensively in the authors' laboratories providing strong evidence that interaction of substrate with residue C221 provides the trigger, and the information is then transmitted along the C221 to H92 to E91 to W412 to G413 pathway to the 4'-amino nitrogen of the thiamin diphosphate cofactor. Earlier, it was found that the C221S substitution reduced the Hill coefficient from 2.0 to 0.8-0.9, the C221A substitution to 1.0, even though C221 is located on the beta domain some 20 A from the active center thiamin diphosphate cofactor, which is at the interface of the alpha and gamma domains. Here are reported experiments on the C221D/C222A and C221E/C222A variants, in which a negative charge is built onto the C221 side chain, to better mimic the effect of a pyruvate molecule covalently bonded to C221 as a thiohemiketal. Both variants were purified to an optimal activity of 70% of the wild-type enzyme, higher activity than that with the earlier uncharged substitutions at this position. The Hill coefficient for both variants is exactly 1.0. The deuterium solvent kinetic isotope effects (SKIE) on k(cat) for these variants were similar to that for the wild-type enzyme and the C221A/C222A variant, suggesting that starting with the first irreversible step (decarboxylation) the rate-limiting transition states are very similar for all of these enzyme forms. In contrast, such SKIE on k(cat)/K(m) are quite different for the C221A/C222A variant (0.62) than for the C221E/C222A or C221D/C222A variants (0.80-0.82), clearly indicating the effect of the C221 substitutions on transition states starting with the binding of the first substrate to the enzyme and terminating with the decarboxylation step. The results provide strong additional evidence for the involvement of residue C221 in the substrate activation process and suggest that the C221D (C221E) substitution shifts the enzyme into a conformation that resembles the activated conformation. A comparison with SKIE for the wild-type enzyme provides insight to changes in hydrogen bonding at the active center as a result of substrate activation.

15N kinetic isotope effects on uncatalyzed and enzymatic deamination of cytidine.

15N isotope effects and solvent deuterium isotope effects have been measured for the hydrolytic deamination of cytidine catalyzed by Escherichia coli cytidine deaminase and for the uncatalyzed reaction proceeding spontaneously in neutral solution at elevated temperatures. The primary (15)(V/K) arising from the exocyclic amino group for wild-type cytidine deaminase acting on its natural substrate, cytidine, is 1.0109 (in H(2)O, pH 7.3), 1.0123 (in H(2)O, pH 4.2), and 1.0086 (in D(2)O, pD 7.3). Increasing solvent D(2)O content has no substantial effect on k(cat) but enhances k(cat)/K(m), with a proton inventory showing that the fractionation factors of at least two protons increase markedly during the reaction. Mutant cytidine deaminases with reduced catalytic activity show more pronounced (15)N isotope effects of 1.0124 (Glu91Ala), 1.0134 (His102Ala), and 1.0158 (His102Asn) at pH 7.3 in H(2)O, as expected for processes in which the chemical transformation of the substrate becomes more rate determining. The isotope effect of mutant His102Asn is 1.033 after correcting for protonation of the -NH(2) group, and represents the intrinsic isotope effect on C-N bond cleavage. This result allows an estimation of the forward commitment of the reaction with the wild-type enzyme. The observed (15)N kinetic isotope effect of the pyrimidine N-3, for wild-type cytidine deaminase acting on cytidine, is 0.9879, which is consistent with protonation and rehybidization of N-3 with hydroxide ion attack on the adjacent carbon to create a tetrahedral intermediate. These results show that enzymatic deamination of cytidine proceeds stepwise through a tetrahedral intermediate with ammonia elimination as the major rate-determining step. The primary (15)N isotope effects observed for the uncatalyzed reaction at pH 7 (1.0021) and pH 12.5 (1.0034) were found to be insensitive to changing temperatures between 100 and 185 degrees C. These results show that the uncatalyzed and the enzymatic deaminations of cytidine proceed by similar mechanisms, although the commitment to C-N bond breaking is greater for the spontaneous reaction.

PH and kinetic isotope effects in d-amino acid oxidase catalysis.

The effects of pH, solvent isotope, and primary isotope replacement on substrate dehydrogenation by Rhodotorula gracilis d-amino acid oxidase were investigated. The rate constant for enzyme-FAD reduction by d-alanine increases approximately fourfold with pH, reflecting apparent pKa values of approximately 6 and approximately 8, and reaches plateaus at high and low pH. Such profiles are observed in all presteady-state and steady-state kinetic experiments, using both d-alanine and d-asparagine as substrates, and are inconsistent with the operation of a base essential to catalysis. A solvent deuterium isotope effect of 3.1 +/- 1.1 is observed on the reaction with d-alanine at pH 6; it decreases to 1.2 +/- 0.2 at pH 10. The primary substrate isotope effect on the reduction rate with [2-D]d-alanine is 9.1 +/- 1.5 at low and 2.3 +/- 0.3 at high pH. At pH 6.0, the solvent isotope effect is 2.9 +/- 0.8 with [2-D]d-alanine, and the primary isotope effect is 8.4 +/- 2.4 in D2O. Thus, primary and solvent kinetic isotope effects (KIEs) are independent of the presence of the other isotope, i.e. the 'double' kinetic isotope effect is the product of the individual KIEs, consistent with a transition state in which rupture of the two bonds of the substrate to hydrogen is concerted. These results support a hydride transfer mechanism for the dehydrogenation reaction in d-amino acid oxidase and argue against the occurrence of any intermediates in the process. A pKa,app of approximately 8 is interpreted to arise from the microscopic ionization of the substrate amino acid alpha-amino group, but also includes contributions from kinetic parameters.

Acid and metal ion promoted hydrolysis of [ N-( o-carboxyphenyl)iminodiacetato](picolinato)chromate(III) complex: a mechanistic study

The sodium salt of the [ N-( o-carboxyphenyl)iminodiacetato]picolinatochromate(III) complex has been synthesised and characterised. In acidic media, the complex has been shown to aquate to diaqua[ N-( o-carboxyphenyl)imminodiacetato]chromium(III) and picolinic acid as the ultimate products. The enhanced kinetic stability exhibited by the aromatic amino acid (cida) may be attributed to (i) resonance interaction of the carboxyl and tertiary nitrogen group with the phenyl ring resulting in a shortening of the bond distance and thus leading to a closer approach of the chelate donor groups, (ii) the presence of a larger number (two five-membered and one six-membered ring) of chelate rings in the ligated amino acid, in comparison to only one five-membered metal–picolinato ring. The variation of the observed pseudo-first-order rate constants with perchloric acid concentration suggests the operation of two concurrent uncatalysed and acid-catalysed pathways. The reaction exhibits a solvent deuterium isotope effect k D/ k H=1.5 at 45 °C. The solvent deuterium isotope effect is consistent with a rapid pre-equilibrium protonation followed by rate-determining ring opening and excludes a mechanism involving concerted attack by [H 3O +]. The metal ions, Cu 2+, Ni 2+, and Zn 2+, promoted hydrolysis in weakly acidic medium has been studied as a function of metal ion concentration and temperature. The metal ion catalysed aquation is proposed to take place through a rapid pre-equilibrium step to form a binuclear complex with the M 2+ ion, followed by the cleavage of the CrO bond in a slow rate-determining step. The catalytic efficiency of the metal ion follows the Irving–Williams order of stability and also parallels the log k M-pic values.

Acid‐catalyzed hydrolysis of bridged bi‐ and tricyclic compounds. XXXVIII—Kinetics and mechanisms of 1‐ and 3‐nortricyclanols

AbstractThe disappearance of 1‐ and 3‐nortricyclanols (1‐OH and 2‐OH) in aqueous perchloric acid was followed by capillary GC at different temperatures and acid concentrations. 1‐OH is ca 1000 times more reactive than 2‐OH. The activation parameters, solvent deuterium isotope effects and parameters of excess acidity equations were measured and the products were studied. Both isomeric nortricyclanols react according to the AdE2 mechanism, i.e. the cyclopropane ring is protonated at the rate‐determining stage of the reaction. The protonation causes, in the case of 1‐OH, an isomerization called homoketonization with 2‐norbornanone as the only product and, in the case of 2‐OH, hydration, i.e. the formation of hydroxyl‐substituted norbornyl cations, the fast attack of which by water produces several norbornanediols. Copyright © 2001 John Wiley &amp; Sons, Ltd.

Characterization of the B12- and iron-sulfur-containing reductive dehalogenase from Desulfitobacterium chlororespirans.

The United Nations and the U.S. Environmental Protection Agency have identified a variety of chlorinated aromatics that constitute a significant health and environmental risk as "priority organic pollutants," the so-called "dirty dozen." Microbes have evolved the ability to utilize chlorinated aromatics as terminal electron acceptors in an energy-generating process called dehalorespiration. In this process, a reductive dehalogenase (CprA), couples the oxidation of an electron donor to the reductive elimination of chloride. We have characterized the B12 and iron-sulfur cluster-containing 3-chloro-4-hydroxybenzoate reductive dehalogenase from Desulfitobacterium chlororespirans. By defining the substrate and inhibitor specificity for the dehalogenase, the enzyme was found to require an hydroxyl group ortho to the halide. Inhibition studies indicate that the hydroxyl group is required for substrate binding. The carboxyl group can be replaced by other functionalities, e.g. acetyl or halide groups, ortho or meta to the chloride to be eliminated. The purified D. chlororespirans enzyme could dechlorinate an hydroxylated PCB (3,3',5,5'-tetrachloro-4,4'-biphenyldiol) at a rate about 1% of that with 3-chloro-4-hydroxybenzoate. Solvent deuterium isotope effect studies indicate that transfer of a single proton is partially rate-limiting in the dehalogenation reaction.

Mycobacterium tuberculosis lipoamide dehydrogenase is encoded by Rv0462 and not by the lpdA or lpdB genes.

The gene encoding dihydrolipoamide dehydrogenase from Mycobacterium tuberculosis, Rv0462, was expressed in Escherichia coli and the protein purified to homogeneity. The 49 kDa polypeptide forms a homodimer containing one tightly bound molecule of FAD/monomer. The results of steady-state kinetic analyses using several reduced pyridine nucleotide analogs and a variety of electron acceptors, and the ability of the enzyme to catalyze the transhydrogenation of NADH and thio-NAD(+) in the absence of D,L-lipoamide, demonstrated that the enzyme uses a ping-pong kinetic mechanism. Primary deuterium kinetic isotope effects on V and V/K at pH 7.5 using NADH deuterated at the C(4)-proS position of the nicotinamide ring are small [(D)(V/K)(NADH) = 1.12 +/- 0.15, (D)V(app) = 1.05 +/- 0.07] when D,L-lipoamide is the oxidant but large and equivalent [(D)(V/K)(NADH) = (D)V = 2.95 +/- 0.03] when 5-hydroxy-1,4-naphthoquinone is the oxidant. Solvent deuterium kinetic isotope effects at pH 5.8, using APADH as the reductant, are inverse with (D)(V/K)(APADH) = 0.73 +/- 0.03, (D)(V/K)(Lip(S))2 = 0.77 +/- 0.03, and (D)V(app) = 0.77 +/- 0.01. Solvent deuterium kinetic isotope effects with 4,4-dithiopyridine (DTP), the 4-thiopyridone product of which requires no protonation, are also inverse with (D)(V/K)(APADH) = 0.75 +/- 0.06, (D)(V/K)(DTP) = 0.71 +/- 0.02, and (D)V(app) = 0.56 +/- 0.15. All proton inventories were linear, indicating that a single proton is being transferred in the solvent isotopically sensitive step. Taken together, these results suggest that (1) the reductive half-reaction (hydride transfer from NADH to FAD) is rate limiting when a quinone is the oxidant, and (2) deprotonation of enzymic thiols, most likely Cys(46) and Cys(41), limits the reductive and oxidative half-reactions, respectively, when D,L-lipoamide is the oxidant.

Transient-State and Steady-State Kinetic Studies of the Mechanism of NADH-Dependent Aldehyde Reduction Catalyzed by Xylose Reductase from the Yeast Candida tenuis

Microbial xylose reductase, a representative aldo-keto reductase of primary sugar metabolism, catalyzes the NAD(P)H-dependent reduction of D-xylose with a turnover number approximately 100 times that of human aldose reductase for the same reaction. To determine the mechanistic basis for that physiologically relevant difference and pinpoint features that are unique to the microbial enzyme among other aldo/keto reductases, we carried out stopped-flow studies with wild-type xylose reductase from the yeast Candida tenuis. Analysis of transient kinetic data for binding of NAD(+) and NADH, and reduction of D-xylose and oxidation of xylitol at pH 7.0 and 25 degrees C provided estimates of rate constants for the following mechanism: E + NADH right arrow over left arrow E.NADH right arrow over left arrow E.NADH + D-xylose right arrow over left arrow E.NADH.D-xylose right arrow over left arrow E.NAD(+).xylitol right arrow over left arrow E.NAD(+) right arrow over left arrow E.NAD(+) right arrow over left arrow E + NAD(+). The net rate constant of dissociation of NAD(+) is approximately 90% rate limiting for k(cat) of D-xylose reduction. It is controlled by the conformational change which precedes nucleotide release and whose rate constant of 40 s(-)(1) is 200 times that of completely rate-limiting E.NADP(+) --> E.NADP(+) step in aldehyde reduction catalyzed by human aldose reductase [Grimshaw, C. E., et al. (1995) Biochemistry 34, 14356-14365]. Hydride transfer from NADH occurs with a rate constant of approximately 170 s(-1). In reverse reaction, the E.NADH --> E.NADH step takes place with a rate constant of 15 s(-1), and the rate constant of ternary-complex interconversion (3.8 s(-1)) largely determines xylitol turnover (0.9 s(-1)). The bound-state equilibrium constant for C. tenuis xylose reductase is estimated to be approximately 45 (=170/3.8), thus greatly favoring aldehyde reduction. Formation of productive complexes, E.NAD(+) and E.NADH, leads to a 7- and 9-fold decrease of dissociation constants of initial binary complexes, respectively, demonstrating that 12-fold differential binding of NADH (K(i) = 16 microM) vs NAD(+) (K(i) = 195 microM) chiefly reflects difference in stabilities of E.NADH and E.NAD(+). Primary deuterium isotope effects on k(cat) and k(cat)/K(xylose) were, respectively, 1.55 +/- 0.09 and 2.09 +/- 0.31 in H(2)O, and 1.26 +/- 0.06 and 1.58 +/- 0.17 in D(2)O. No deuterium solvent isotope effect on k(cat)/K(xylose) was observed. When deuteration of coenzyme selectively slowed the hydride transfer step, (D)()2(O)(k(cat)/K(xylose)) was inverse (0.89 +/- 0.14). The isotope effect data suggest a chemical mechanism of carbonyl reduction by xylose reductase in which transfer of hydride ion is a partially rate-limiting step and precedes the proton-transfer step.

Stopped-flow kinetic study of the peroxidase reactions of mangano-microperoxidase-8

We have investigated the kinetics for the peroxidase-type reaction of mangano microperoxidase 8 (Mn(III)-MP8) by the time-resolved and single-wavelength stopped-flow technique. The formation of intermediate and its subsequent reaction with substrates were studied separately. Oxidation of Mn(III)-MP8 by H2O2 at pH 10.7 yields an intermediate (1) with a rate constant of 2.9 x10(4) M-1 s-1. The formation of 1 exhibits no deuterium solvent isotope effect, favoring the homolytic cleavage of the Mn(III)-MP8 bound hydroperoxide. The rate for the formation of 1 increases sharply as the pH increases and no other intermediate was detected in the entire pH range. Addition of substrate to 1 leads to the regeneration of Mn(III)-MP8. Monitoring the conversion of 1 to Mn(III)-MP8 allows the determination of the substrate reactivity. The substrate reactivity varies by more than two orders of magnitude ranging from 1.04 x 10(6) M-1 s-1 for ascorbic acid to 4.61 x 10(3) M-1s-1 for aniline. It is linearly correlated with the reduction potential for most of the substrates studied, with the easier oxidized species showing greater reactivity. The substrate reactivity drops rapidly as the pH increases. The substrate reactivity at pH 10.7 for the Mn(III)-MP8 system is smaller than that of the corresponding Fe(III)-MP8 system by 2- to 25-fold, depending on the substrate used.

Effect of an E461G Mutation of β-Galactosidase (Escherichia coli, lac Z) on pL Rate Profiles and Solvent Deuterium Isotope Effects

An E461G mutation of β-galactosidase results in the disappearance of the high pL (L = H, D) downward break in the rate profiles for kcat/Km for wild-type enzyme-catalyzed hydrolysis of 4-nitrophenyl β-d-galactopyranoside (Gal-OPNP) and a decrease from (kcat)HOH/(kcat)DOD = 1.7 to (kcat)HOH/(kcat)DOD = 1.2 in the solvent deuterium isotope effect. These observations provide evidence that the propionic acid side chain of Glu 461 is protonated at catalytically active free β-galactosidase and they are consistent with a role for this residue in Brønsted acid catalysis at the leaving group. The earlier observation that this same E461G mutation results in the loss of a downward break at high pH in the rate profile for ks for transfer of the β-d-galactopyranosyl group from β-galactosidase to water cannot be simply explained by a mechanism in which the single side chain of Glu 461 functions to provide general acid catalysis in the rate limiting step for formation of the β-d-galactopyranosyl intermediate and general base catalysis of breakdown of this intermediate. Evidence is presented that there may be different catalytic mechanisms, with different roles for the side chain for Glu-461, for nucleophilic addition of water and of small alkyl alcohols to the β-d-galactopyranosyl reaction intermediate.