Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization.

Glycolate oxidase from spinach has been expressed in Saccharomyces cerevisiae. The active enzyme was purified to near-homogeneity (purification factor approximately 1400-fold) by means of hydroxyapatite and anion-exchange chromatography. The purified glycolate oxidase is nonfluorescent and has absorbance peaks at 448 (epsilon = 9200 M-1 cm-1) and 346 nm in 0.1 M phosphate buffer, pH 8.3. The large bathochromic shift of the near-UV band indicates that the N(3) position is deprotonated at pH 8.3. A pH titration revealed that the pK of the N(3) is shifted from 10.3 in free flavin to 6.4 in glycolate oxidase. Glycolate oxidase is competitively inhibited by oxalate with a Kd of 0.24 mM at 4 degrees C in 0.1 M phosphate buffer, pH 8.3. Three pieces of evidence demonstrate that glycolate oxidase stabilizes a negative charge at the N(1)-C(2 = O) locus: the enzyme forms a tight sulfite complex with a Kd of 2.7 x 10(-7) M and stabilizes the anionic flavosemiquinone and the benzoquinoid form of 8-mercapto-FMN. Steady-state analysis at pH 8.3, 4 degrees C, yielded a Km = 1 x 10(-3) M for glycolate and Km = 2.1 x 10(-4) M for oxygen. The turnover number has been determined to be 20 s-1. Stopped-flow studies of the reductive (k = 25 s-1) and oxidative (k = 8.5 x 10(4) M-1 s-1) half-reactions have identified the reduction of glycolate oxidase to be the rate-limiting step.     

Coenzyme A dithioesters: synthesis, characterization and reaction with citrate synthase and acetyl-CoA:choline O-acetyltransferase

Acyl dithioesters of CoA have been synthesized by transesterification. The alpha-hydrogens have a spectrally determined pKa of 12.5 +/- 0.14. The hydroxide catalyzed enolization rate is estimated to be 600 M-1.s-1. The absorbance of the dithioester, lambda max = 306 nm, can be used to monitor both the condensation and transesterification reactions that use CoA-Ac as a substrate. For citrate synthase at pH 7.4 Vmax = (4.0 +/- 0.4).10(-4) s-1 and Km = 53 +/- 7.5 microM, which are 2.10(-6) and 3.3-times the Vmax and Km values observed for CoAS-Ac, while for Ac-CoA: choline O-acetyltransferase (EC 2.3.1.6) at pH 7.0 Vmax = (1.1 +/- 0.2).10(-2) mumol.s-1.(mg protein)-1 and Km = 83 +/- 33 microM, which are 0.077 and 10-times the values observed with CoAS-Ac, respectively. The CoA dithioesters are stable at low pH, but hydrolyze with a second-order rate constant of 8.2.10(-2) M-1.s-1 at pH 11.4. The spectral properties of these dithioesters should allow these analogs to be used as probes of the structure of enzyme bound intermediates.     

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

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

Isotope effect studies of the role of metal ions in isocitrate dehydrogenase.

Pig heart NADP+-dependent isocitrate dehydrogenase requires a metal ion for activity. Under optimum conditions (pH 7.5, Mg2+ present), the carbon isotope effect is k12/k13 = 0.9989 +/- 0.0004 for the carboxyl carbon undergoing decarboxylation and hydrogen isotope effects are VmaxH/VmaxD = 1.09 +/- 0.04 and (Vmax/Km)H/(Vmax/Km)D = 0.76 +/- 0.12 with threo-D,L-[2-2H]isocitric acid. Deuterium isotope effects measured by the equilibrium perturbation technique under the same conditions are VH/VD = 1.20 for the forward reaction and 1.02 for the reverse reaction. Under these conditions the rate-determining step in the enzymatic reaction must be product release. Dissociation of isocitrate from the enzyme-isocitrate complex and the enzyme-NADP+ complex must be two or more orders of magnitude slower than the chemical steps. The catalytic activity of the enzyme is about tenfold lower in the presence of Ni2+ than in the presence of Mg2+. The carbon isotope effect in the presence of Ni2+ at pH 7.5 is k12/k13 = 1.0051 +/- 0.0012 and the hydrogen isotope effects are VmaxH/VmaxD = 0.98 +/- 0.07 and (Vmax/Km)H/(Vmax/Km)D = 1.11 +/- 0.14. Thus, the rate decrease caused by substitution of Ni2+ for Mg2+ must result from the effects of metal on substrate and product binding and dissociation, rather than effects of metal on catalysis. However, a more detailed analysis of the carbon isotope effects reveals that there is also a large metal effect on the rate of the decarboxylation step, consistent with the view that the carbonyl oxygen of the oxalosuccinate intermediate is coordinated to the metal during decarboxylation.     

The effect of glycerol on the activity of beta-glucosidase from Botryodiplodia theobromae Pat.

1. In the activity of the high-Mr beta-glucosidase A (beta-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. on o-nitrophenyl beta-D-glucopyranoside as substrate, both Vmax. and Km increased non-linearly with increasing concentration of glycerol, and the Vmax./Km(app.) ratio decreased non-linearly with increasing concentration of glycerol. 2. No increase in rate was observed with phenyl beta-D-glucopyranoside as substrate in the presence of up to 250 mM-glycerol, indicating that glucosylation is rate-limiting with this substrate. 3. With o-nitrophenyl beta-D-glucopyranoside, p-nitrophenyl beta-D-glucopyranoside and phenyl beta-D-glucopyranoside as substrates, kappa cat. values of 793.7 s-1, 62.8 s-1 and 5.4 s-1 respectively were calculated. 4. With o-nitrophenyl beta-D-glucopyranoside and phenyl beta-D-glucopyranoside as substrate, alpha-deuterium kinetic isotope effects of 1.9 +/- 0.03 and 1.01 +/- 0.01 respectively were found; in the presence of 200 mM-glycerol the values were 1.21 +/- 0.03 and 1.02 +/- 0.01 respectively. 5. In the presence of a large excess of o-nitrophenyl beta-D-glucopyranoside [( S] = 35.7 Km), the amount of o-nitrophenol and also of the transglucosylation product formed by beta-glucosidase action increased non-linearly, whereas that of glucose formed decreased non-linearly with increasing glycerol concentration. 6. All these results were found to fit the data calculated from rate equations derived on the basis of the proposed mechanism of enzyme action involving two ion-pair intermediates and a covalent alpha-D-glucosyl-enzyme in the reaction sequence [Umezurike (1987) Biochem. J. 241, 455-462].     

The kinetic mechanism of beef kidney D-aspartate oxidase

The mechanism of action of the flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been investigated by steady-state and stopped flow kinetic studies using D-aspartate and O2 as substrates in 50 mM KPi, 0.3 mM EDTA, pH 7.4, 4 degrees C. Steady-state results indicate that a ternary complex containing enzyme, O2, and substrate (or product) is an obligatory intermediate in catalysis. The kinetic parameters are turnover number = 11.1 s-1, Km(D-Asp) = 2.2 x 10(-3) M, Km(O2) = 1.7 x 10(-4) M. Rapid reaction studies show that 1) the reductive half reaction is essentially irreversible with a maximum rate of reduction of 180 s-1; 2) the free reduced enzyme cannot be the species which is reoxidized during turnover since its reoxidation by oxygen (second order rate constant equal to 5.3 x 10(2) M-1 s-1) is too slow to be of relevance in catalysis; 3) reduced enzyme can bind a ligand rapidly and be reoxidized as a complex at a rate faster than that observed for the free reduced enzyme; 4) the rate of reoxidation of reduced enzyme by oxygen during turnover is dependent on both O2 and D-aspartate concentrations (second order rate constant of reaction between O2 and reduced enzyme-substrate complex equal to 6.2 x 10(4) M-1 s-1); and 5) the rate-limiting step in catalysis occurs after reoxidation of the enzyme and before its reduction in the following turnover. A mechanism involving reduction of enzyme by substrate, dissociation of product from reduced enzyme, binding of a second molecule of substrate to the reduced enzyme, and reoxidation of the reduced enzyme-substrate complex is proposed for the enzyme-catalyzed oxidation of D-aspartate.     

Mechanism of O2 activation by cytochrome P450cam studied by isotope effects and transient state kinetics

The early steps in dioxygen activation by the monooxygenase cytochrome P450cam (CYP101) include binding of O2 to ferrous P450cam to yield the ferric-superoxo form (oxyP450cam) followed by an irreversible, long-range electron transfer from putidaredoxin to reduce the oxyP450cam. The steady state kinetic parameter kcat/Km(O2) has been studied by a variety of probes that indicate a small D2O solvent isotope effect (1.21 +/- 0.08), a very small solvent viscosogen effect, and a 16O/18O isotope effect of 1.0147 +/- 0.0007. This latter value, which can be compared with the 16O/18O equilibrium isotope effect of 1.0048 +/- 0.0003 measured for oxyP450cam formation, is attributed to a primarily rate-limiting outer-sphere electron transfer from the heme iron center as O2 that has prebound to protein approaches the active site cofactor. The electron transfer from putidaredoxin to oxyP450cam was investigated by rapid mixing at 25 degrees C to complement previous lower-temperature measurements. A rate of 390 +/- 23 s-1 (and a near-unity solvent isotope effect) supports the view that the long-range electron transfer from reduced putidaredoxin to oxyP450cam is rapid relative to dissociation of O2 from the enzyme. P450cam represents the first enzymatic reaction of O2 in which both equilibrium and kinetic 16O/18O isotope effects have been measured.     

One-proton catalysis by the alpha-L-arabinofuranosidase III of Monilinia fructigena.

1. Michaelis-Menten parameters for the hydrolysis of p-nitrophenyl alpha-L-arabinofuranoside were measured as a function of pL (pH or pD) in both 1H2O and 2H2O. 2. The variation of both Vmax. and Vmax./Km with pL is sigmoid, the pK governing Vmax. shifting from 6.34 +/- 0.05 in 1H2O to 6.84 +/- 0.07 in 2H2O, and that governing Vmax./Km from 5.89 +/- 0.03 in 1H2O to 6.38 +/- 0.05 in 2H2O. 3. In the plateau regions there is a small inverse solvent isotope effect on Vmax./Km (0.92), and one of 1.45 on Vmax. 4. The variation of Vmax. with isotopic composition is strictly linear, indicating that the isotope effect arises from the transfer of a single proton.     

Evidence that both protium and deuterium undergo significant tunneling in the reaction catalyzed by bovine serum amine oxidase

The magnitudes of primary and secondary H/T and D/T kinetic isotope effects have been measured in the bovine serum amine oxidase catalyzed oxidation of benzylamine from 0 to 45 degrees C. Secondary H/T and D/T kinetic effects are small and in the range anticipated from equilibrium isotope effects; Arrhenius preexponential factors (AH/AT and AD/AT) determined from the temperature dependence of isotope effects also indicate semiclassical behavior. By contrast, primary H/T and D/T isotope effects, 35.2 +/- 0.8 and 3.07 +/- 0.07, respectively, at 25 degrees C, are larger than semiclassical values and give anomalously low preexponential factor ratios, AH/AT = 0.12 +/- 0.04 and AD/AT = 0.51 +/- 0.10. Stopped-flow studies indicate similar isotope effects on cofactor reduction as seen in the steady state, consistent with a single rate-limiting C-H bond cleavage step for Vmax/Km. The comparison of primary and secondary isotope effects allows us to rule out appreciable coupling between the primary and secondary hydrogens at C-1 of the substrate. From the properties of primary isotope effects, we conclude that both protium and deuterium undergo significant tunneling in the course of substrate oxidation. These findings represent the first example of quantum mechanical effects in an enzyme-catalyzed proton abstraction reaction.     

Limits of diffusion in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta.

The catalytic mechanism for the enzymatic hydrolysis of a series of paraoxon analogues by the phosphotriesterase from Pseudomonas diminuta has been determined. The Br?nsted plots relating the pKa of the leaving group to the observed kinetic parameters, Vmax and V/Km, are both nonlinear. This observation is consistent with a change in the rate-limiting step from chemical to physical events as the pKa of the leaving group is decreased. This conclusion is confirmed by the effects of solvent viscosity on Vmax and V/Km for the same series of analogues. The data were fitted to the scheme E k1A in equilibrium k2 EA k3----EP k7----E'P k9----E + products where EA is the enzyme-substrate complex, EP is the enzyme-product complex, E'P is the enzyme-product complex after a viscosity-independent unimolecular reaction, and the values for k1, k2, k7, and k9 are 4.1 X 10(7) M-1 s-1, 2550 s-1, 3370 s-1, and 5940 s-1, respectively. The magnitude of the chemical step, represented by k3, is dependent on the pKa of the leaving group phenol as predicted by the Br?nsted equation (log k3 = beta pKa + C) where beta = -1.8 and the constant (C) = 17.7. The magnitude of beta indicates that the transition state for substrate hydrolysis is very product-like.     

Nucleoside hydrolase from Crithidia fasciculata. Metabolic role, purification, specificity, and kinetic mechanism

Crithidia fasciculata cells grown on complex medium with added [8-14C, 5'-3H]inosine or [8-14C,5'-3H]adenosine metabolize greater than 50% of the salvaged nucleosides through a pathway involving N-glycoside bond cleavage. Cell extracts contain a substantial nucleoside hydrolase activity but an insignificant purine nucleoside phosphorylase. The nucleoside hydrolase has been purified 1000-fold to greater than 99% homogeneity from kilogram quantities of C. fasciculata. The enzyme is a tetramer of Mr 34,000 subunits to give an apparent holoenzyme Mr of 143,000 by gel filtration. All of the commonly occurring nucleosides are substrates. The Km values vary from 0.38 to 4.7 mM with purine nucleosides binding more tightly than the pyrimidines. Values of Vmax/Km vary from 3.4 x 10(3) M-1 s-1 to 1.7 x 10(5) M-1 s-1 with the pyrimidine nucleosides giving the larger values. The turnover rate for inosine is 32 s-1 at 30 degrees C. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The hydrolytic reaction can be reversed to give an experimental Keq of 106 M with H2O taken as unity. The product dissociation constants for ribose and hypoxanthine are 0.7 and 6.2 mM, respectively. Deoxynucleosides or 5'-substituted nucleosides are poor substrates or do not react, and are poor inhibitors of the enzyme. The enzyme discriminates against methanol attack from solvent during steady-state catalysis, indicating the participation of an enzyme-directed water nucleophile. The pH profile for inosine hydrolysis gives two apparent pKa values of 6.1 with decreasing Vmax/Km values below the pKa and a plateau at higher pH values. These effects are due to the pH sensitivity of the Vmax values, since Km is independent of pH. The pH profile implicates two negatively charged groups which stabilize a transition state with oxycarbonium character.     

Transport of glucose and fructose in rat hepatocytes at 37 degrees C

The kinetic parameters for transport of the nonmetabolizable glucose analogue 3-O-methyl-D-glucose and the relationship between transport and metabolism of D-glucose and D-fructose were determined in isolated rat hepatocytes at 37 degrees C and pH 7.4. 3-O-Methylglucose at a very low concentration (0.1 mM) equilibrated with the intracellular water with a rate constant of 0.41 s-1. Km for equilibrium exchange entry was 5.5 mM and Vmax was 2.2 mM X s-1 and similar results were obtained when using the zero-trans entry protocol. The rate constant for entry of tracer D-glucose was 0.15 s-1 and Km for glucose was about 20 mM. The phosphorylation rate for D-glucose was much slower than the transport rate. The rate constant for D-fructose entry was about 0.04 s-1, the apparent Km was about 100 mM and Vmax about 5 mM X s-1. The concentration dependence of 3-O-methylglucose inhibition of labelled fructose transport revealed biphasic kinetics indicating that fructose was transferred by both the glucose transporter and a fructose transporter. At concentrations lower than 1 mM, fructose metabolism appeared to be limited by the transport step.     

Deuterium isotope effects in the unusual addition of toluene to fumarate catalyzed by benzylsuccinate synthase

The first step in the anaerobic metabolism of toluene is a highly unusual reaction: the addition of toluene across the double bond of fumarate to produce (R)-benzylsuccinate, which is catalyzed by benzylsuccinate synthase. Benzylsuccinate synthase is a member of the glycyl radical-containing family of enzymes, and the reaction is initiated by abstraction of a hydrogen atom from the methyl group of toluene. To gain insight into the free energy profile of this reaction, we have measured the kinetic isotope effects on Vmax and Vmax/Km when deuterated toluene is the substrate. At 30 degrees C the isotope effects are 1.7 +/- 0.2 and 2.9 +/- 0.1 on Vmax and Vmax/Km, respectively; at 4 degrees C they increase slightly to 2.2 +/- 0.2 and 3.1 +/- 0.1, respectively. We compare these results with the theoretical isotope effects on Vmax and Vmax/Km that are predicted from the free energy profile for the uncatalyzed reaction, which has previously been computed using density functional theory [Himo, F. (2002) J. Phys. Chem. B 106, 7688-7692]. The comparison allows us to draw some conclusions on how the enzyme may catalyze this unusual reaction.     

Isotopic probes yield microscopic constants: separation of binding energy from catalytic efficiency in the bovine plasma amine oxidase reaction

Bovine plasma amine oxidase catalyzes the oxidative deamination of primary amines. The reaction can be viewed as two half-reactions: enzyme reduction by substrate followed by enzyme reoxidation by dioxygen. Anaerobic stopped-flow kinetic measurements of the first half-reaction indicate very large deuterium isotope effects for benzylamine, m-tyramine, and dopamine, Dk = 13.5 +/- 1.3, which are ascribed to an intrinsic isotope effect. From the insensitivity of these isotope effects to amine concentration, stopped-flow data provide substrate dissociation constants, K1, and rate constants for the C-H bond cleavage step, k3, directly. Steady-state isotope effects have also been measured for benzylamine and six ring-substituted phenethylamines. Whereas a small range of values for kcat, 0.38-1.2 s-1, and Dkcat, 5.4-8.8, is observed, kcat/Km = 1.3 X 10(2) to 3.8 X 10(4) M-1 S-1 and D(kcat/Km) = 5.6-16.1 indicate a marked effect of ring substituent. As described earlier [Miller, S., & Klinman, J.P. (1982) Methods Enzymol. 87, 711], the availability of an intrinsic isotope effect for an enzymatic reaction permits calculation of microscopic constants from steady-state data. By employment of a minimal mechanism for bovine plasma amine oxidase involving a single precatalytic and multiple postcatalytic enzyme-substrate complexes, equations have been derived that allow calculation of k3 and K1 when DKeq congruent to 1 less than Dk. Unexpectedly, in the case of K1, we have shown that this parameter can be calculated from steady-state parameters without the requirement for an intrinsic isotope effect. This result should have general application to both ping-pong and sequential ternary-complex enzyme mechanisms. Of significance for future applications of steady-state isotope effects to the calculation of microscopic constants, values for K1 and k3 derived from steady-state parameters and single turnover measurements indicate excellent agreement. Compilation of parameters among six ring-substituted phenethylamines reveals alteration in delta G for enzyme-substrate complex formation by 2.8 kcal/mol, together with an essentially invariant rate constant for C-H bond activation. A detailed discussion of the relevance of these findings to the interrelationship of binding energy and catalytic efficiency in enzyme reactions is presented.     

Determination of glucose oxidase oxidation-reduction potentials and the oxygen reactivity of fully reduced and semiquinoid forms.

The oxidation-reduction potential values for the two electron transfers to glucose oxidase were obtained at pH 5.3, where the neutral radical is the stable form, and at pH 9.3, where the anion radical is the stable form. The midpoint potentials at 25 degrees were: pH 5.3 EFl1ox + e- H+ equilibrium EFlH. Em1 = -0.063 +/- 0.011 V EFlH. + e- + H+ equilibrium EFlredH2 Em2 = -0.065 +/- 0.007 V pH 9.3 EFlox + e- EFi- Em1 = -0.200 +/- 0.010 V EFi- + e- + H+ equilibrium EFlredH- Em2 = -0.240 +/- 0.005 V All potentials were measured versus the standard hydrogen electrode (SHE). The potentials indicated that glucose oxidase radicals are stabilized by kinetic factors and not by thermodynamic energy barriers. The pK for the glucose oxidase radical was 7.28 from dead time stopped flow measurements and the extinction coefficient of the neutral semiquinone was 4140 M-1 cm-1 at 570 nm. Both radical forms reacted with oxygen in a second order fashion. The rate at 25 degrees for the neutral semiquinone was 1.4 X 10(4) M-1 s-1; that for the anion radical was 3.5 X 10(4) M-1 s-1. The rate of oxidation of the neutral radical changed by a factor of 9 for a temperature difference of 22 degrees. For the anion radical, the oxidation rate changed by a factor of 6 for a 22 degrees change in temperature. We studied the oxygen reactivity of the 2-electron reduced form of the enzyme over a wide wavelength range and failed to detect either oxygenated flavin derivatives or semiquinoid forms as intermediates. The rate of reoxidation of fully reduced glucose oxidase at pH 9.3 was dependent on ionic strength.     

The inhibition of yeast enolase by Li+ and Na+1

The activity of yeast enolase is inhibited by Li+ and Na+. At pH 7.1, inhibition by Li+ is "mixed" with respect to Mg2+; both Vmax and Km (Mg2+) are increased by Li+. The inhibition by Li+ appears to be partial, indicating that enzyme with Li+ bound is active. The step inhibited by Li+ cannot be proton abstraction since Li+ decreases the kinetic isotope effect on Vmax. At pH 9.2, where proton abstraction is no longer partially rate-limiting, inhibiton by Li+ is competitive with respect to Mg2+. The rate of enzyme-catalyzed exchange of the C-2 hydrogen with solvent is not affected by Li+. We interpret these results as follows: Li+ (and Na+) binds to enolase and decreases the rate of at least one step in the mechanism. At pH 7.1, this step is partially rate-limiting; at pH 9.2, this step is a fast step in the reaction. The step inhibited by Li+ cannot be proton abstraction but may be release of product (phosphoenol pyruvate) or Mg2+.     

Comparison of the reaction progress of calcineurin with Mn2+ and Mg2+

Activation of calcineurin by Mn2+ and Mg2+ was compared using a heavy atom isotope analogue of the substrate p-nitrophenyl phosphate (pNPP). Heavy atom isotope effects were measured for Mg2+ activation and compared to published results of the isotope effects with Mn2+ as the activating metal. Isotope effects were measured for the kinetic parameter Vmax/Km at the nonbridging oxygen atoms [18(V/K)nonbridge]; at the position of bond cleavage in the bridging oxygen atom [18(V/K)bridge]; and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The isotope effects increased in magnitude upon changing from an optimal pH to a nonoptimal pH; the 18(V/K)bridge effect increased from 1.0154 (+/-0.0007) to 1.0198 (+/-0.0002), and the 15(V/K) effect increased from 1.0018 (+/-0. 0002) to 1.0021 (+/-0.0003). The value for 18(V/K)nonbridge is 0. 9910 (+/-0.0003) at pH 7.0. As with Mn2+, the 18(V/K)nonbridge isotope effect indicated that the dianion was the substrate for catalysis, and that a dissociative transition state was operative for the phosphoryl transfer. Comparison to results for Mn2+ activation suggested that chemistry was more rate-limiting with Mg2+ than with Mn2+. Changing the activating metal concentration showed opposite trends with increasing Mg2+ increasing the commitment factor and seemingly making the chemistry less rate-limiting. The influence of viscosity was evaluated as well to gauge the role of chemistry. The activation of calcineurin-catalyzed hydrolysis of pNPP1 by Mg2+ or Mn2+ at pH 7.0 was compared in the presence of viscogens, glycerol and poly(ethylene glycol). Increasing glycerol caused different effects with the two activators. With Mn2+ as the activator, calcineurin activity showed a normal response with kcat and kcat/Km decreasing with viscosity. There was an inverse response with Mg2+ as the activator as values of kcat/Km increased with viscosity. From values of the normalized kcat/Km with Mn2+, the chemistry was found to be partially rate-limiting, consistent with previous heavy atom isotope studies (22). The effect observed for Mg2+ seems consistent with a change in the rate-limiting step for the two different metals at pH 7.0.     

Tegumental glucose permeability in male and female Schistosoma mansoni

Tegumental hexose transporters have been kinetically characterized in mated and separated male and female Schistosoma mansoni 8-12 wk postinfection. Significant gender-specific differences in Km and Vmax were observed. In mated males, the estimated constants (mean +/- SE) were: Km = 0.63 +/- 0.31 mM, Vmax = 0.93 +/- 0.44 nmol/mg worm water/min, and the Kd = 0.25 +/- 0.09 microliter/mg worm water/min. In mated females the kinetics were: Km = 0.99 +/- 0.40 mM, Vmax = 1.22 +/- 0.42 nmol/mg worm water/min, and Kd = 0.60 +/- 0.14 microliter/mg worm water/min. The influx of 2-deoxy-D-glucose and 3-O-methylglucose has been similarly characterized; these analogs share the same glucose transporter in male and female schistosomes. 2-Deoxy-D-glucose has a higher affinity, and 3-O-methylglucose a lower affinity, than does glucose. Because mated male schistosomes supply glucose to female partners, similarities between the free glucose concentration of the male and the affinity of the transporter determined for mated female schistosomes suggest that male-to-female transfer may be a potentially rate-limiting step in glucose utilization by the female. Permeability x surface are (PS) products and Vmax/Km ratios were significantly elevated in mated schistosomes, suggesting that the transporter is primarily localized to the dorsal surface of the male. Gender- and mating-specific analyses of PS products indicate that tegumental permeability to glucose is significantly increased in mated schistosomes, and compares very favorably to that of the host liver.     

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.     

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.