Electron transfer within xanthine oxidase: a solvent kinetic isotope effect study

Solvent kinetic isotope effect studies of electron transfer within xanthine oxidase have been performed, using a stopped-flow pH-jump technique to perturb the distribution of reducing equivalents within partially reduced enzyme and follow the kinetics of reequilibration spectrophotometrically. It is found that the rate constant for electron transfer between the flavin and one of the iron-sulfur centers of the enzyme observed when the pH is jumped from 10 to 6 decreases from 173 to 25 s-1 on going from H2O to D2O, giving an observed solvent kinetic isotope effect of 6.9. An effect of comparable magnitude is observed for the pH jump in the opposite direction, the rate constant decreasing from 395 to 56 s-1. The solvent kinetic isotope effect on kobs is found to be directly proportional to the mole fraction of D2O in the reaction mix for the pH jump in each direction, consistent with the effect arising from a single exchangeable proton. Calculations of the microscopic rate constants for electron transfer between the flavin and the iron-sulfur center indicate that the intrinsic solvent kinetic isotope effect for electron transfer from the neutral flavin semiquinone to the iron-sulfur center designated Fe/S I is substantially greater than for electron transfer in the opposite direction and that the observed solvent kinetic isotope effect is a weighted averaged of the intrinsic isotope effects for the forward and reverse microscopic electron-transfer steps.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Chemical mechanism and rate-limiting steps in the reaction catalyzed by Streptococcus faecalis NADH peroxidase

The pH dependence of the kinetic parameters V, V/KNADH, and V/KH2O2 has been determined for the flavoenzyme NADH peroxidase. Both V/KNADH and V/KH2O2 decrease as groups exhibiting pK's of 9.2 and 9.9, respectively, are deprotonated. The V profile decreases by a factor of 5 as a group exhibiting a pK of 7.2 is deprotonated. Primary deuterium kinetic isotope effects on NADH oxidation are observed on V only, and the magnitude of DV is independent of H2O2 concentration at pH 7.5. DV/KNADH is pH independent and equal to 1.0 between pH 6 and pH 9.5, but DV is pH dependent, decreasing from a value of 7.2 at pH 5.5 to 1.9 at pH 9.5. The shape of the DV versus pH profile parallels that observed in the V profile and yields a similar pK of 6.6 for the group whose deprotonation decreases DV. Solvent kinetic isotope effects obtained with NADH or reduced nicotinamide hypoxanthine dinucleotide as the variable substrate are observed on V only, while equivalent solvent kinetic isotope effects on V and V/K are observed when H2O2 is used as the variable substrate. In all cases linear proton inventories are observed. Primary deuterium kinetic isotope effects on V for NADH oxidation decrease as the solvent isotopic composition is changed from H2O to D2O. These data are consistent with a change in the rate-limiting step from a step in the reductive half-reaction at low pH to a step in the oxidative half-reaction at high pH. Analysis of the multiple kinetic isotope effect data suggests that at high D2O concentrations the rate of a single proton transfer step in the oxidative half-reaction is slowed. These data are used to propose a chemical mechanism involving the pH-dependent protonation of a flavin hydroxide anion, following flavin peroxide bond cleavage.     

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

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

Deuterium kinetic isotope effects in the p-side pathway for quinol oxidation by the cytochrome b(6)f complex.

Plastoquinol oxidation and proton transfer by the cytochrome b(6) f complex on the lumen side of the chloroplast thylakoid membrane are mediated by high and low potential electron transport chains. The rate constant for reduction, k(bred), of cytochrome b(6) in the low potential chain at ambient pH 7.5-8 was twice that, k(fred), of cytochrome f in the high potential chain, as previously reported. k(bred) and k(fred) have a similar pH dependence in the presence of nigericin/nonactin, decreasing by factors of 2.5 and 4, respectively, from pH 8 to an ambient pH = 6, close to the lumen pH under conditions of steady-state photosynthesis. A substantial kinetic isotope effect, k(H2O)/k(D2O), was found over the pH range 6-8 for the reduction of cytochromes b(6) and f, and for the electrochromic band shift associated with charge transfer across the b(6)f complex, showing that isotope exchange affects the pK values linked to rate-limiting steps of proton transfer. The kinetic isotope effect, k(bred)(H2O)/k(bred) (D2O) approximately 3, for reduction of cytochrome b in the low potential chain was approximately constant from pH 6-8. However, the isotope effect for reduction of cytochrome f in the high potential chain undergoes a pH-dependent transition below pH 6.5 and increased 2-fold in the physiological region of the lumen pH, pH 5.7-6.3, where k(fred)(H2O)/k(fred)(D2O) approximately 4. It is proposed that a rate-limiting step for proton transfer in the high potential chain resides in the conserved, buried, and extended water chain of cytochrome f, which provides the exit port for transfer of the second proton derived from p-side quinol oxidation and a "dielectric well" for charge balance.     

Solvent and solvent proton dependent steps in the galactose oxidase reaction

Solvent and solvent proton dependent steps involved in the mechanism of the enzyme galactose oxidase have been examined. The deuterium kinetic solvent isotope effect (KSIE) on the velocity of the galactose oxidase catalyzed oxidation of methyl beta-galactopyranoside by O2 was measured. Examination of the thermodynamic activation parameters for the reaction indicated that the isotope effect was attributable to a slightly less favorable delta H value, consistent with a KSIE on proton transfer. A detailed kinetic analysis was performed, examining the effect of D2O on the rate of reaction over the pH range 4.8-8.0. Both pL-rate profiles exhibited bell-shaped curves. Substitution of D2O as solvent shifted the pKes values for the enzymic central complex: pKes1 from 6.30 to 6.80 and pKes2 from 7.16 to 7.35. Analysis of the observed shifts in dissociation constants was performed with regard to potential hydrogenic sites. pKes1 can be attributed to a histidine imidazole, while pKes2 is tentatively assigned to a Cu2+-bound water molecule. A proton inventory was performed (KSIE = +1.55); the plot of kcat vs. mole fraction D2O was linear, indicating the existence of a single solvent-derived proton involved in a galactose oxidase rate-determining step (or steps). The pH dependence of CN- inhibition was also examined. The Ki-pH profile indicated that a group ionization, with pKa = 7.17, modulated CN- inhibition; Ki was at a minimum when this group was in the protonated state. The inhibition profile followed the alkaline limit of the pH-rate profile for the enzymic reaction, suggesting that the group displaced by CN- was also deprotonating above pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solvent isotope effects for lipoprotein lipase catalyzed hydrolysis of water-soluble p-nitrophenyl esters

Solvent deuterium isotope effects on the rates of lipoprotein lipase (LpL) catalyzed hydrolysis of the water-soluble esters p-nitrophenyl acetate (PNPA) and p-nitrophenyl butyrate (PNPB) have been measured and fall in the range 1.5-2.2. The isotope effects are independent of substrate concentration, LpL stability, and reaction temperature and hence are effects on chemical catalysis and not due to a medium effect of D2O on LpL stability and/or conformation. pL (L = H or D) vs. rate profiles for the Vmax/Km of LpL-catalyzed hydrolysis of PNPB increase sigmoidally with increasing pL. Least-squares analysis of the profiles gives pKaH2O = 7.10 +/- 0.01, pKaD2O = 7.795 +/- 0.007, and a solvent isotope effect on limiting velocity at high pL of 1.97 +/- 0.03. Because the pL-rate profiles are for the Vmax/Km of hydrolysis of a water-soluble substrate, the measured pKa's are intrinsic acid-base ionization constants for a catalytically involved LpL active-site amino acid side chain. Benzeneboronic acid, a potent inhibitor of LpL-catalyzed hydrolysis of triacylglycerols [Vainio, P., Virtanen, J. A., & Kinnunen, P. K. J. (1982) Biochim. Biophys. Acta 711, 386-390], inhibits LpL-catalyzed hydrolysis of PNPB, with Ki = 6.9 microM at pH 7.36, 25 degrees C. This result and the solvent isotope effects for LpL-catalyzed hydrolysis of water-soluble esters are interpreted in terms of a proton transfer mechanism that is similar in many respects to that of the serine proteases.     

L-ascorbic acid quenching of singlet delta molecular oxygen in aqueous media: generalized antioxidant property of vitamin C

L-ascorbic acid quenches singlet (1 delta g) molecular oxygen in aqueous media (pH 6.8 for [1H]H2O and pD 7.2 for [2H]D2O) as measured directly by monitoring (0,0) 1 delta g leads to 3 sigma-g emission at 1.28 micron. Singlet oxygen was generated at room temperature in the solutions via photosensitization of sodium chrysene sulfonate; this sulfonated polycyclic hydrocarbon was synthesized to provide a water soluble chromophore inert to usual dye-ascorbate photobleaching. A marked isotope effect is found; kHQ2O is 3.3 times faster than kDQ2O, suggesting ascorbic acid is chemically quenching singlet oxygen.     

13C and 15N isotope effects as a probe of the chemical mechanism of Escherichia coli aspartate transcarbamylase.

13C and 15N isotope effects have been measured for the aspartate transcarbamylase (ATCase) reaction in an effort to elucidate the chemical mechanism of this highly regulated enzyme. The observed 15(V/K(asp))H2O value for the ATCase holoenzyme at saturing carbamyl phosphate and 12 mM L-aspartate is 1.0045 at pH 7.5, and this value remains unchanged in the presence of 5 mM ATP (activator) or 5 mM CTP (inhibitor). The fact that the isotope effect is not changed by the allosteric modifiers supports the conclusion that the kinetic properties of the active form of ATCase are not influenced by ATP or CTP. The observed 15(V/K(asp)) values for the catalytic subunit of ATCase are also the same as those determined for the holoenzyme, suggesting that the chemical mechanisms of both enzyme species are the same. Quantitative analysis of 13C and 15N isotope effects in both H2O and D2O has led to the proposal of a chemical model for the ATCase reaction which involves a precatalytic conformational change to form an activated complex that facilitates deprotonation of L-aspartate by an enzyme functional group. Nucleophilic attack on the carbonyl carbon of carbamyl phosphate by the alpha-amino group of L-aspartate results in the formation of a tetrahedral intermediate. An intramolecular proton transfer leads to formation of products N-carbamyl-L-aspartate and inorganic phosphate.     

Solvent-isotope and pH effects on flagellar rotation in Escherichia coli

We studied changes in speed of the flagellar rotary motor of Escherichia coli when tethered cells or cells carrying small latex spheres on flagellar stubs were shifted from H(2)O to D(2)O or subjected to changes in external pH. In the high-torque, low-speed regime, solvent isotope effects were found to be small; in the low-torque, high-speed regime, they were large. The boundaries between these regimes were close to those found earlier in measurements of the torque-speed relationship of the flagellar rotary motor (, Biophys. J. 65:2201-2216;, Biophys. J., 78:1036-1041). This observation provides direct evidence that the decline in torque at high speed is due primarily to limits in rates of proton transfer. However, variations of speed (and torque) with shifts of external pH (from 4.7 to 8.8) were small for both regimes. Therefore, rates of proton transfer are not very dependent on external pH.     

Comparison of metal-ion-dependent cleavages of RNA by a DNA enzyme and a hammerhead ribozyme

Joyce's DNA enzyme catalyzes cleavage of RNAs with almost the same efficiency as the hammerhead ribozyme. The cleavage activity of the DNA enzyme was pH dependent, and the logarithm of the cleavage rate increased linearly with pH from pH 6 to pH 9 with a slope of approximately unity. The existence of an apparent solvent isotope effect, with cleavage of RNA by the DNA enzyme in H(2)O being 4.3 times faster than cleavage in D(2)O, was in accord with the interpretation that, at a given pH, the concentration of the active species (deprotonated species) is 4.3 times higher in H(2)O than the concentration in D(2)O. This leads to the intrinsic isotope effect of unity, demonstrating that no proton transfer occurs in the transition state in reactions catalyzed by the DNA enzyme. Addition of La(3+) ions to the Mg(2+)-background reaction mixture inhibited the DNA enzyme-catalyzed reactions, suggesting the replacement of catalytically and/or structurally important Mg(2+) ions by La(3+) ions. Similar kinetic features of DNA enzyme mediated cleavage of RNA and of hammerhead ribozyme-mediated cleavage suggest that a very similar catalytic mechanism is used by the two types of enzyme, despite their different compositions.     

Kinetic and chemical mechanism of the dihydrofolate reductase from Mycobacterium tuberculosis

Dihydrofolate reductase from Mycobacterium tuberculosis (MtDHFR) catalyzes the NAD(P)-dependent reduction of dihydrofolate, yielding NAD(P)(+) and tetrahydrofolate, the primary one-carbon unit carrier in biology. Tetrahydrofolate needs to be recycled so that reactions involved in dTMP synthesis and purine metabolism are maintained. In this work, we report the kinetic characterization of the MtDHFR. This enzyme has a sequential steady-state random kinetic mechanism, probably with a preferred pathway with NADPH binding first. A pK(a) value for an enzymic acid of approximately 7.0 was identified from the pH dependence of V, and the analysis of the primary kinetic isotope effects revealed that the hydride transfer step is at least partly rate-limiting throughout the pH range analyzed. Additionally, solvent and multiple kinetic isotope effects were determined and analyzed, and equilibrium isotope effects were measured on the equilibrium constant. (D(2)O)V and (D(2)O)V/K([4R-4-(2)H]NADH) were slightly inverse at pH 6.0, and inverse values for (D(2)O)V([4R-4-(2)H]NADH) and (D(2)O)V/K([4R-4-(2)H]NADH) suggested that a pre-equilibrium protonation is occurring before the hydride transfer step, indicating a stepwise mechanism for proton and hydride transfer. The same value was obtained for (D)k(H) at pH 5.5 and 7.5, reaffirming the rate-limiting nature of the hydride transfer step. A chemical mechanism is proposed on the basis of the results obtained here.     

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

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

Solvent isotope effects and the pH dependence of laccase activity under steady-state conditions

Solvent isotope effects and the pH dependence of laccase catalysis under steady-state conditions were examined with a rapid reductant to assess the potential roles of protein protic groups and the catalytic mechanism. The pH dependence of both reductant-dependent and reductant-independent steps showed bell-shaped profiles implicating at least two protic groups in each case. The apparent pKa values were: for the reductant-independent step(s), pK alpha 1 = 8.98 +/- 0.02 and pK alpha 2 = 5.91 +/- 0.03; for the reductant-dependent step(s), pK' alpha 1 = 7.55 +/- 0.12, pK' alpha 2 = 8.40 +/- 0.23. No solvent isotope effect on reductant-dependent steps was detected other than a standard shift effect. However, a significant solvent isotope effect on a reductant-independent step(s) was observed; kH/kD = 2.12 at the pH optimum of 7.5. The concentration dependence of the D2O effect indicated that a single proton was involved. Simulations of the p(H,D) data suggested that the solvent isotope effect was associated with the protein protic group required in its undissociated form (pK alpha 2). The pH effects on reductant-dependent steps are apparently associated with reductant-dependent steps that occur between O2 binding and water formation in the catalytic reaction sequence.     

Solvent isotope effects on the reaction catalyzed by yeast hexokinase

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

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

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

Hydrogen peroxide induces the dissociation of GroEL into monomers that can facilitate the reactivation of oxidatively inactivated rhodanese

Although, several studies have been reported on the effects of oxidants on the structure and function of other molecular chaperones, no reports have been made so far for the chaperonin GroEL. The ability of GroEL to function under oxidative stress was investigated in this report by monitoring the effects of hydrogen peroxide (H(2)O(2)) on the structure and refolding activity of this protein. Using fluorescence spectroscopy and light scattering, we observed that GroEL showed increases in exposed hydrophobic sites and changes in tertiary and quaternary structure. Differential sedimentation, gel electrophoresis, and circular dichroism showed that H(2)O(2) treated GroEL underwent irreversible dissociation into monomers with partial loss of secondary structure. Relative to other proteins, GroEL was found to be highly resistant to oxidative damage. Interestingly, GroEL monomers produced under these conditions can facilitate the reactivation of H(2)O(2)-inactivated rhodanese but not urea-denatured rhodanese. Recovery of approximately 84% active rhodanese was obtained with either native or oxidized GroEL in the absence of GroES or ATP. In comparison, urea-denatured GroEL, BSA and the refolding mixture in the absence of proteins resulted in the recovery of 72, 50, and 49% rhodanese activity, respectively. Previous studies have shown that GroEL monomers can reactivate rhodanese. Here, we show that oxidized monomeric GroEL can reactivate oxidized rhodanese suggesting that GroEL retains the ability to protect proteins during oxidative stress.     

Backbone dynamics of a model membrane protein: measurement of individual amide hydrogen-exchange rates in detergent-solubilized M13 coat protein using 13C NMR hydrogen/deuterium isotope shifts

Hydrogen-exchange rates have been measured for individual assigned amide protons in M13 coat protein, a 50-residue integral membrane protein, using a 13C nuclear magnetic resonance (NMR) equilibrium isotope shift technique. The locations of the more rapidly exchanging amides have been determined. In D2O solutions, a peptide carbonyl resonance undergoes a small upfield isotope shift (0.08-0.09 ppm) from its position in H2O solutions; in 1:1 H2O/D2O mixtures, the carbonyl line shape is determined by the exchange rate at the adjacent nitrogen atom. M13 coat protein was labeled biosynthetically with 13C at the peptide carbonyls of alanine, glycine, phenylalanine, proline, and lysine, and the exchange rates of 12 assigned amide protons in the hydrophilic regions were measured as a function of pH by using the isotope shift method. This equilibrium technique is sensitive to the more rapidly exchanging protons which are difficult to measure by classical exchange-out experiments. In proteins, structural factors, notably H bonding, can decrease the exchange rate of an amide proton by many orders of magnitude from that observed in the freely exposed amides of model peptides such as poly(DL-alanine). With corrections for sequence-related inductive effects [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158], the retardation of amide exchange in sodium dodecyl sulfate solubilized coat protein has been calculated with respect to poly(DL-alanine). The most rapidly exchanging protons, which are retarded very little or not at all, are shown to occur at the N- and C-termini of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tritium effect in peroxidation of ehtanol by liver catalase.

1. Simultaneous determination of the rate of appearance of 3H in water from [(1R)-1-3H1] ethanol and the rate of acetaldehyde formation in the presence of rat or ox liver catalase under conditions of steady-state generation of H2O2 allowed calculation of the 3H isotope effect. The mean value of 2.52 obtained for rat liver catalase at 37 degrees C and pH 6.3-7.7 was independent of both ethanol concentration and the rate of H2O2 generation over a wide range. At 25 degrees C a slightly lower mean value of 2.40 was obtained with the ox liver catalase. 2. Neither the product, acetaldehyde, nor 4-methylpyrazole influenced the two rates measured in the assay. 3. Relating the value obtained for the 3H isotope effect to a known value for the 2H isotope effect strongly supports the view that both values are close to the true isotope effect with the respective substituted compounds on the rate constant in the catalytic step involving scission of the C-H bond. 4. The constancy of the isotope effect under various conditions makes it possible to use it for interpretations in vivo. 5. It was established that beta-D-galactose dehydrogenase exhibits B-specificity towards the nicotinamide ring in NAD.