Examining the relative timing of hydrogen abstraction steps during NAD(+)-dependent oxidation of secondary alcohols catalyzed by long-chain D-mannitol dehydrogenase from Pseudomonas fluorescens using pH and kinetic isotope effects

Mannitol dehydrogenases (MDH) are a family of Zn(2+)-independent long-chain alcohol dehydrogenases that catalyze the regiospecific NAD(+)-dependent oxidation of a secondary alcohol group in polyol substrates. pH and primary deuterium kinetic isotope effects on kinetic parameters for reaction of recombinant MDH from Pseudomonas fluorescens with D-mannitol have been measured in H(2)O and D(2)O at 25 degrees C and used to determine the relative timing of C-H and O-H bond cleavage steps during alcohol conversion. The enzymatic rates decreased at low pH; apparent pK values for log(k(cat)/K(mannitol)) and log k(cat) were 9.2 and 7.7 in H(2)O, respectively, and both were shifted by +0.4 pH units in D(2)O. Proton inventory plots for k(cat) and k(cat)/K(mannitol) were determined at pL 10.0 using protio or deuterio alcohol and were linear at the 95% confidence level. They revealed the independence of primary deuterium isotope effects on the atom fraction of deuterium in a mixed H(2)O-D(2)O solvent and yielded single-site transition-state fractionation factors of 0.43 +/- 0.05 and 0.47 +/- 0.01 for k(cat)/K(mannitol) and k(cat), respectively. (D)(k(cat)/K(mannitol)) was constant (1.80 +/- 0.20) in the pH range 6.0-9.5 and decreased at high pH to a limiting value of approximately 1. Measurement of (D)(k(cat)/K(fructose)) at pH 10.0 and 10.5 using NADH deuterium-labeled in the 4-pro-S position gave a value of 0.83, the equilibrium isotope effect on carbonyl group reduction. A mechanism of D-mannitol oxidation by MDH is supported by the data in which the partly rate-limiting transition state of hydride transfer is stabilized by a single solvation catalytic proton bridge. The chemical reaction involves a pH-dependent internal equilibrium which takes place prior to C-H bond cleavage and in which proton transfer from the reactive OH to the enzyme catalytic base may occur. Loss of a proton from the enzyme at high pH irreversibly locks the ternary complex with either alcohol or alkoxide bound in a conformation committed of undergoing NAD(+) reduction at a rate about 2.3-fold slower than the corresponding reaction rate of the protonated complex. Transient kinetic studies for D-mannitol oxidation at pH(D) 10.0 showed that the solvent isotope effect on steady-state turnover originates from a net rate constant of NADH release that is approximately 85% rate-limiting for k(cat) and 2-fold smaller in D(2)O than in H(2)O.     

Localized control of proton transfer through the D-pathway in cytochrome c oxidase: application of the proton-inventory technique

In the reaction cycle of cytochrome c oxidase from Rhodobacter sphaeroides, one of the steps that are coupled to proton pumping, the oxo-ferryl-to-oxidized transition (F --> O), displays a large kinetic deuterium isotope effect of about 7. In this study we have investigated in detail the dependence of the kinetics of this reaction step ?k(FO)(chi) on the fraction (chi) D(2)O in the enzyme solution (proton-inventory technique). According to a simplified version of the Gross-Butler equation, from the shape of the graph describing k(FO)(chi)/k(FO)(0), conclusions can be drawn concerning the number of protonatable sites involved in the rate-limiting proton-transfer reaction step. Even though the proton-transfer reaction during the F --> O transition takes place over a distance of at least 30 A and involves a large number of protonatable sites, the proton-inventory analysis displayed a linear dependence, which indicates that the entire deuterium isotope effect of 7 is associated with a single protonatable site. On the basis of experiments with site-directed mutants of cytochrome c oxidase, this localized proton-transfer rate control is proposed to be associated with glutamate (I-286) in the D-pathway. Consequently, the results indicate that proton transfer from the glutamate controls the rate of all events during the F --> O reaction step. The proton-inventory analysis of the overall enzyme turnover reveals a nonlinear plot characteristic of at least two protonatable sites involved in the rate-limiting step in the transition state, which indicates that this step does not involve proton transfer through the same pathway (or through the same mechanism) as during the F --> O transition.     

New insights on the proton pump associated with cytochrome b6f turnovers from the study of H/D substitution effects on the electrogenicity and electron transfer reactions

We have studied the effect of protium/deuterium substitution on different kinetics associated with the turnovers of cytochrome b(6)f complex in whole cells of Chlamydomonas reinhardtii. Both the oxidation of cytochrome f and the reduction of hemes b were only little affected by the isotopic substitution. Contrasting with this, the initial slope of the electrogenic phase associated with cytochrome b(6)f turnover was slowed by a factor of 4 by H(2)O/D(2)O substitution. Whereas in the presence of H(2)O the electrogenic phase developed concomitantly with cytochrome b reduction, it lagged for a few hundreds of microseconds after cytochrome b reduction in the presence of D(2)O. We propose that a proton pump is triggered by the oxidation of plastoquinol at the Q(o) site. The proton transfer is specifically delayed upon isotopic substitution, accounting for the lack of significant effect on the electron-transfer reaction as well as for the strong decrease of the initial rate of the electrogenic phase.     

Reaction of ascorbic acid with cytochrome b561. Concerted electron and proton transfer.

Rate constants for reduction of cytochrome b561 by internal ascorbate (k0A) and oxidation by external ferricyanide (k1F) were determined as a function of pH from rates of steady-state electron transfer across chromaffin-vesicle membranes. The pH dependence of electron transfer from cytochrome b561 to ferricyanide (k1F) may be attributed to the pH dependence of the membrane surface potential. The rate constant for reduction by internal ascorbate (k0A), like the previously measured rate constant for reduction by external ascorbate (k-1A), is not very pH-dependent and is not consistent with reduction of cytochrome b561 by the ascorbate dianion. The rate at which ascorbate reduces cytochrome b561 is orders of magnitude faster than the rate at which it reduces cytochrome c, despite the fact that midpoint reduction potentials favor reduction of cytochrome c. Moreover, the rate constant for oxidation of cytochrome b561 by ferricyanide (k1F) is smaller than the previously measured rate constant for oxidation by semidehydroascorbate, despite the fact that ferricyanide has a higher midpoint reduction potential. These results may be reconciled by a mechanism in which electron transfer between cytochrome b561 and ascorbate/semidehydroascorbate is accelerated by concerted transfer of a proton. This may be a general property of biologically significant electron transfer reactions of ascorbic acid.     

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.     

Electron transfer from the Rieske iron-sulfur protein (ISP) to cytochrome f in vitro. Is a guided trajectory of the ISP necessary for competent docking?

The time course of electron transfer in vitro between soluble domains of the Rieske iron-sulfur protein (ISP) and cytochrome f subunits of the cytochrome b(6)f complex of oxygenic photosynthesis was measured by stopped-flow mixing. The domains were derived from Chlamydomonas reinhardtii and expressed in Escherichia coli. The expressed 142-residue soluble ISP apoprotein was reconstituted with the [2Fe-2S] cluster. The second-order rate constant, k(2)((ISP-f)) = 1.5 x 10(6) m(-1) s(-1), for ISP to cytochrome f electron transfer was <10(-2) of the rate constant at low ionic strength, k(2)((f-PC))(> 200 x 10(6) m(-1) s(-1)), for the reduction of plastocyanin by cytochrome f, and approximately 1/30 of k(2)((f-PC)) at the ionic strength estimated for the thylakoid interior. In contrast to k(2)((f-PC)), k(2)((ISP-f)) was independent of pH and ionic strength, implying no significant role of electrostatic interactions. Effective pK values of 6.2 and 8.3, respectively, of oxidized and reduced ISP were derived from the pH dependence of the amplitude of cytochrome f reduction. The first-order rate constant, k(1)((ISP-f)), predicted from k(2)((ISP-f)) is approximately 10 and approximately 150 times smaller than the millisecond and microsecond phases of cytochrome f reduction observed in vivo. It is proposed that in the absence of electrostatic guidance, a productive docking geometry for fast electron transfer is imposed by the guided trajectory of the ISP extrinsic domain. The requirement of a specific electrically neutral docking configuration for ISP electron transfer is consistent with structure data for the related cytochrome bc(1) complex.     

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.     

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.     

Interconversion of Cu(I) and Cu(II) forms of galactose oxidase: comparison of reduction potentials

A procedure for the preparation of the fully reduced Cu(I) form of galactose oxidase, GOase(red), involving reduction of GOase(semi) (or GOase(ox)) with non-coordinating [Ru(NH(3))(6)](2+) (51 mV vs. nhe) is described. Air-free conditions and a two-fold excess of [Ru(NH(3))(6)](2+) give a stable product with no further UV-Vis changes over >1.5 h. Rate constants for the reduction of GOase(semi) (k(f)=860 M(-1) s(-1)) give a first-order [H(+)]-dependence (pK(1a)=7.9), but the reverse process involving [Ru(NH(3))(6)](3+) oxidation of GOase(red) (k(b)=18.6 M(-1) s(-1)) is independent of pH (5.5 to 9.5). The reduction potential E(2)(o)' (vs. nhe) for the GOase(semi)/GOase(red) (i.e. Cu(II)/Cu(I)) couple is 149 mV at pH 7.5, which varies from 160 mV (pH 5.5) to 120 mV (pH 10.5), suggesting pK(1a) (GOase(semi)) and pK(2a) (GOase(red)) acid dissociation constants both involving Tyr-495. It is concluded that pK(2a) is for acid dissociation of uncoordinated H(+)Tyr-495. Consistent with this interpretation rate constants/M(-1) s(-1) for the GOase(semi) Tyr495 Phe variant, k(f)=1.59x10(3) and k(b)=16.1, respectively, are independent of pH and give a reduction potential of 169 mV. Comparisons are made of reduction potentials (E(1)(o)'/mV pH 7.5) for the GOase(ox)/GOase(semi) (i.e. Tyr(.)/Tyr) couple, and are for the Cys228Gly variant (630), for enzyme with N(3)(-) for H(2)O at the substrate binding exogenous site (393), and for apo-protein (570). These compare with previously reported values for the variants Trp290His (730) and Tyr495Phe (450), and together serve to quantify different contributions to the unusually small E(1)(o)' of 400 mV for the Tyr(.)/Tyr couple. At pH 7.5 the reduction potential for the two-equivalent GOase(ox)/GOase(red) couple is calculated to be 275 mV. The rate constant for the reaction of GOase(red) with GOase(ox) is 4.4x10(3) M(-1) s(-1) at pH 7.5.     

Redox-coupled proton pumping activity in cytochrome b6f,as evidenced by the pH dependence of electron transfer in whole cells of Chlamydomonas reinhardtii

The pH dependence of cytochrome b(6)f catalytic activity has been measured in whole cells of the green alga Chlamydomonas reinhardtii over the 5-8 range. An acid pH slowed the reactions occurring at the lumenal side of the complex (cytochrome b(6) and f reduction) and affected also the rate and amplitude of the slow electrogenic reaction (phase b), which is supposed to reflect transmembrane electron flow in the complex. On the other hand, a direct measurement of the transmembrane electron flow from the kinetics of cytochrome b(6) oxidation revealed no pH sensitivity. This suggests that a substantial fraction of the electrogenicity associated with cytochrome b(6)f catalysis is not due to electron transfer in the b(6) hemes but to a plastoquinol-oxidation-triggered charge movement, in agreement with previous suggestions that a redox-coupled proton pump operates in cytochrome b(6)f complex. The pH dependence of cytochrome b(6)f activity has also been measured in two mutant strains, where the glutamic 78 of the conserved PEWY sequence of subunit IV has been substituted for a basic (E78K) and a polar (E78Q) residue [Zito, F., Finazzi, G., Joliot, P., and Wollman, F.-A. (1998) Biochemistry 37, 10395-10403]. Their comparison with the wild type revealed that this residue plays an essential role in plastoquinol oxidation at low pH, while it is not required for efficient activity at neutral pH. Its involvement in gating the redox-coupled proton pumping activity is also shown.     

Solvent deuterium isotope effect on the oxidation of o-diphenols by tyrosinase

A solvent deuterium isotope effect on the catalytic affinity (K(m)) and rate constant (k(cat)) of tyrosinase in its action on 4-tert-butylcatechol (TBC) was observed. Both parameters decreased as the molar fraction of deuterated water in the medium increased, while the k(cat)/K(m) ratio remained constant. In a proton inventory study, the representation of k(cat)(f(n))/k(cat)(f(0)) and K(m)(f(n))/K(m)(f(0)) vs. n (atom fractions of deuterium) was linear, indicating that, of the four protons transferred from the two molecules of substrate and which are oxidized in one turnover, only one is responsible for the isotope effects. The fractionation factor of 0.64+/-0.02 contributed to identifying the possible proton acceptor. Possible mechanistic implications are discussed.     

Kinetic and spectroscopic characterization of the gamma-carbonic anhydrase from the methanoarchaeon Methanosarcina thermophila.

The zinc and cobalt forms of the prototypic gamma-carbonic anhydrase from Methanosarcina thermophila were characterized by extended X-ray absorption fine structure (EXAFS) and the kinetics were investigated using steady-state spectrophotometric and (18)O exchange equilibrium assays. EXAFS results indicate that cobalt isomorphously replaces zinc and that the metals coordinate three histidines and two or three water molecules. The efficiency of either Zn-Cam or Co-Cam for CO(2) hydration (k(cat)/K(m)) was severalfold greater than HCO(3-) dehydration at physiological pH values, a result consistent with the proposed physiological function for Cam during growth on acetate. For both Zn- and Co-Cam, the steady-state parameter k(cat) for CO(2) hydration was pH-dependent with a pK(a) of 6.5-6.8, whereas k(cat)/K(m) was dependent on two ionizations with pK(a) values of 6.7-6.9 and 8.2-8.4. The (18)O exchange assay also identified two ionizable groups in the pH profile of k(cat)/K(m) with apparent pK(a) values of 6.0 and 8.1. The steady-state parameter k(cat) (CO(2) hydration) is buffer-dependent in a saturable manner at pH 8. 2, and the kinetic analysis suggested a ping-pong mechanism in which buffer is the second substrate. The calculated rate constant for intermolecular proton transfer is 3 x 10(7) M(-1) s(-1). At saturating buffer concentrations and pH 8.5, k(cat) is 2.6-fold higher in H(2)O than in D(2)O, suggesting that an intramolecular proton transfer step is at least partially rate-determining. At high pH (pH > 8), k(cat)/K(m) is not dependent on buffer and no solvent hydrogen isotope effect was observed, consistent with a zinc hydroxide mechanism. Therefore, at high pH the catalytic mechanism of Cam appears to resemble that of human CAII, despite significant structural differences in the active sites of these two unrelated enzymes.     

The onset of the deuterium isotope effect in cytochrome c oxidase

We have investigated the dynamics of proton equilibration within the proton-transfer pathway of cytochrome c oxidase from bovine heart that is used for the transfer of both substrate and pumped protons during reaction of the reduced enzyme with oxygen (D-pathway). The kinetics of the slowest phase in the oxidation of the enzyme (the oxo-ferryl --> oxidized transition, F --> O), which is associated with proton uptake, were studied by monitoring absorbance changes at 445 nm. The rate constant of this transition, which is 800 s(-)(1) in H(2)O (at pH approximately 7.5), displayed a kinetic deuterium isotope effect of approximately 4 (i.e., the rate was approximately 200 s(-)(1) in 100% D(2)O). To investigate the kinetics of the onset of the deuterium isotope effect, fully reduced, solubilized CO-bound cytochrome c oxidase in H(2)O was mixed rapidly at a ratio of 1:5 with a D(2)O buffer saturated with oxygen. After a well-defined time period, CO was flashed off using a short laser flash. The time between mixing and flashing off CO was varied within the range 0. 04-10 s. The results show that for the bovine enzyme, the onset of the deuterium isotope effect takes place within two time windows of O transition is internal proton transfer from a site, proposed to be Glu (I-286) (R. sphaeroides amino acid residue numbering), to the binuclear center. The spontaneous equilibration of protons/deuterons with this site in the interior of the protein is slow (approximately 1 s).     

Role of active site histidines in the two half-reactions of the aryl-alcohol oxidase catalytic cycle

The crystal structure of aryl-alcohol oxidase (AAO), a flavoenzyme involved in lignin degradation, reveals two active-site histidines, whose role in the two enzyme half-reactions was investigated. The redox state of flavin during turnover of the variants obtained show a stronger histidine involvement in the reductive than in the oxidative half-reaction. This was confirmed by the k(cat)/K(m(Al)) and reduction constants that are 2-3 orders of magnitude decreased for the His546 variants and up to 5 orders for the His502 variants, while the corresponding O(2) constants only decreased up to 1 order of magnitude. These results confirm His502 as the catalytic base in the AAO reductive half-reaction. The solvent kinetic isotope effect (KIE) revealed that hydroxyl proton abstraction is partially limiting the reaction, while the α-deuterated alcohol KIE showed a stereoselective hydride transfer. Concerning the oxidative half-reaction, directed mutagenesis and computational simulations indicate that only His502 is involved. Quantum mechanical/molecular mechanical (QM/MM) reveals an initial partial electron transfer from the reduced FADH(-) to O(2), without formation of a flavin-hydroperoxide intermediate. Reaction follows with a nearly barrierless His502H(+) proton transfer that decreases the triplet/singlet gap. Spin inversion and second electron transfer, concomitant with a slower proton transfer from flavin N5, yields H(2)O(2). No solvent KIE was found for O(2) reduction confirming that the His502 proton transfer does not limit the oxidative half-reaction. However, the small KIE on k(cat)/K(m(Ox)), during steady-state oxidation of α-deuterated alcohol, suggests that the second proton transfer from N5H is partially limiting, as predicted by the QM/MM simulations.     

A cytochrome c encoded by the nar operon is required for the synthesis of active respiratory nitrate reductase in Thermus thermophilus

The pgr1 mutant of Arabidopsis thaliana carries a single point mutation (P194L) in the Rieske subunit of the cytochrome b6/f (cyt b6/f) complex and is characterised by a reduced electron transport activity at saturating light intensities in vivo. We have investigated the electron transport in this mutant under in vitro conditions. Measurements of P700 reduction kinetics and of photosynthetic electron transport rates indicated that electron transfer from cyt b6/f to photosystem I is not generally reduced in the mutant, but that the pH dependence of this reaction is altered. The data imply that the pH-dependent inactivation of electron transport through cyt b6/f is shifted by about 1 pH unit to more alkaline pH values in pgr1 thylakoids in comparison with wild-type thylakoids. This interpretation was confirmed by determination of the transmembrane deltapH at different stromal pH values showing that the lumen pH in pgr1 mutant plants cannot drop below pH 6 reflecting most likely a shift of the pK and/or the redox potential of the oxidised Rieske protein.     

Kinetic study of the catalytic mechanism of mannitol dehydrogenase from Pseudomonas fluorescens.

To characterize catalysis by NAD-dependent long-chain mannitol 2-dehydrogenases (MDHs), the recombinant wild-type MDH from Pseudomonas fluorescens was overexpressed in Escherichia coli and purified. The enzyme is a functional monomer of 54 kDa, which does not contain Zn(2+) and has B-type stereospecificity with respect to hydride transfer from NADH. Analysis of initial velocity patterns together with product and substrate inhibition patterns and comparison of primary deuterium isotope effects on the apparent kinetic parameters, (D)k(cat), (D)(k(cat)/K(NADH)), and (D)(k(cat)/K(fructose)), show that MDH has an ordered kinetic mechanism at pH 8.2 in which NADH adds before D-fructose, and D-mannitol and NAD are released in that order. Isomerization of E-NAD to a form which interacts with D-mannitol nonproductively or dissociation of NAD from the binary complex after isomerization is the slowest step (>/=110 s(-)(1)) in D-fructose reduction at pH 8.2. Release of NADH from E-NADH (32 s(-)(1)) is the major rate-limiting step in mannitol oxidation at this pH. At the pH optimum for D-fructose reduction (pH 7.0), the rate of hydride transfer contributes significantly to rate limitation of the catalytic cascade and the overall reaction. (D)(k(cat)/K(fructose)) decreases from 2.57 at pH 7.0 to a value of 相似文献   

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.     

Staphylococcus aureus sortase transpeptidase SrtA: insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism

The Staphylococcus aureus transpeptidase SrtA catalyzes the covalent attachment of LPXTG-containing virulence and colonization-associated proteins to cell-wall peptidoglycan in Gram-positive bacteria. Recent structural characterizations of staphylococcal SrtA, and related transpeptidases SrtB from S. aureus and Bacillus anthracis, provide many details regarding the active site environment, yet raise questions with regard to the nature of catalysis and active site cysteine thiol activation. Here we re-evaluate the kinetic mechanism of SrtA and shed light on aspects of its catalytic mechanism. Using steady-state, pre-steady-state, bisubstrate kinetic studies, and high-resolution electrospray mass spectrometry, revised steady-state kinetic parameters and a ping-pong hydrolytic shunt kinetic mechanism were determined for recombinant SrtA. The pH dependencies of kinetic parameters k(cat)/K(m) and k(cat) for the substrate Abz-LPETG-Dap(Dnp)-NH(2) were bell-shaped with pK(a) values of 6.3 +/- 0.2 and 9.4 +/- 0.2 for k(cat) and 6.2 +/- 0.2 and 9.4 +/- 0.2 for k(cat)/K(m). Solvent isotope effect (SIE) measurements revealed inverse behavior, with a (D)2(O)k(cat) of 0.89 +/- 0.01 and a (D)2(O)(k(cat)/K(m)) of 0.57 +/- 0.03 reflecting an equilibrium SIE. In addition, SIE measurements strongly implicated Cys184 participation in the isotope-sensitive rate-determining chemical step when considered in conjunction with an inverse linear proton inventory for k(cat). Last, the pH dependence of SrtA inactivation by iodoacetamide revealed a single ionization for inactivation. These studies collectively provide compelling evidence for a reverse protonation mechanism where a small fraction (ca. 0.06%) of SrtA is competent for catalysis at physiological pH, yet is highly active with an estimated k(cat)/K(m) of >10(5) M(-)(1) s(-)(1).     

Sulfide-dependent photosynthetic electron flow coupled to proton translocation in thylakoids of the cyanobacterium Oscillatoria limnetica

Light-induced proton translocation coupled to sulfide-dependent electron transport has been studied in isolated thylakoids of the cyanobacterium Oscillatoria limnetica. The thylakoids are obtained by osmotic shock of washed spheroplasts, prepared with glycine-betaine as the osmotic stabilizer. 13C NMR studies suggests that betaine is the major osmoregulator in O. limnetica. Thylakoid preparations obtained from both sulfide-induced anoxygenic cells and noninduced oxygenic cells are capable of proton pumping coupled to phenazinemethosulfate-mediated cyclic electron flow. However, only in the induced thylakoids can sulfide-dependent proton gradient (delta pH) formation be measured, using either NADP or methyl viologen as the terminal acceptor. Sulfide-dependent delta pH formation correlates with a high-affinity electron donation site (apparent Km 44 microM at pH 7.9). This site is not lost upon washing of the thylakoids. In addition, both sulfide-dependent electron transport and delta pH formation are sensitive to inhibitors of the cytochrome b6f complex such as 2-n-nonyl-4-hydroxyquinoline-N-oxide, 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol, or stigmatellin. Sulfide-dependent NADP photoreduction of low affinity (which does not saturate by as much as 7 mM sulfide) is detected in both induced and noninduced thylakoids, but this activity is insensitive to the inhibitors and is not coupled to proton transport. It is suggested that the adaptation of O. limnetica to anoxygenic photosynthesis involves the induction of a thylakoid factor(s) which creates a high-affinity site for sulfide, and the transfer of its electrons via the cytochrome b6f complex, coupled to proton translocation.     

Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant

Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member of the aldo-keto reductase (AKR) superfamily of proteins and enzymes. Active site His-113 is conserved among AKRs, but a unified mechanism of how it affects catalytic activity is outstanding. We have replaced His-113 by alanine using site-directed mutagenesis, determined a 2.2 A structure of H113A mutant bound to NADP(+), and compared catalytic reaction profiles of NADH-dependent reduction of different aldehydes catalyzed by the wild type and the mutant. Deuterium kinetic isotope effects (KIEs) on k(cat) and k(cat)/K(m xylose) show that, relative to the wild type, the hydride transfer rate constant (k(7) approximately 0.16 s(-1)) has decreased about 1000-fold in H113A whereas xylose binding was not strongly affected. No solvent isotope effect was seen on k(cat) and k(cat)/K(m xylose) for H113A, suggesting that proton transfer has not become rate-limiting as a result of the mutation. The pH profiles of log(k(cat)/K(m xylose)) for the wild type and H113A decreased above apparent pK(a) values of 8.85 and 7.63, respectively. The DeltapK(a) of -1.2 pH units likely reflects a proximally disruptive character of the mutation, affecting the position of Asp-50. A steady-state kinetic analysis for H113A-catalyzed reduction of a homologous series of meta-substituted benzaldehyde derivatives was carried out, and quantitative structure-reactivity correlations were used to factor the observed kinetic substituent effect on k(cat) and k(cat)/K(m aldehyde) into an electronic effect and bonding effects (which are lacking in the wild type). Using the Hammett sigma scale, electronic parameter coefficients (rho) of +0.64 (k(cat)) and +0.78 (k(cat)/K(m aldehyde)) were calculated and clearly differ from rho(k(cat)/K(aldehyde)) and rho(k(cat)) values of +1.67 and approximately 0.0, respectively, for the wild-type enzyme. Hydride transfer rate constants of H113A, calculated from kinetic parameters and KIE data, display a substituent dependence not seen in the corresponding wild-type enzyme rate constants. An enzymic mechanism is proposed in which His-113, through a hydrogen bond from Nepsilon2 to aldehyde O1, assists in catalysis by optimizing the C=O bond charge separation and orbital alignment in the ternary complex.