Structure-activity relationships in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta

The mechanism and substrate specificity of the phosphotriesterase from Pseudomonas diminuta have been examined. The enzyme hydrolyzes a large number of phosphotriester substrates in addition to paraoxon (diethyl p-nitrophenyl phosphate) and its thiophosphate analogue, parathion. The two ethyl groups in paraoxon can be changed to propyl and butyl groups, but the maximal velocity and Km values decrease substantially. The enzyme will not hydrolyze phosphomonoesters or -diesters. There is a linear correlation between enzymatic activity and the pKa of the phenolic leaving group for 16 paraoxon analogues. The beta value in the corresponding Br?nsted plot is -0.8. No effect on either Vmax or Vmax/Km is observed when sucrose is used to increase the relative solvent viscosity by 3-fold. These results are consistent with rate-limiting phosphorus-oxygen bond cleavage. A plot of log V versus pH for the hydrolysis of paraoxon shows one enzymatic group that must be unprotonated for activity with a pKa of 6.1. The deuterium isotope effect by D2O on Vmax and Vmax/Km is 2.4 and 1.2, respectively, and the proton inventory is linear, which indicates that only one proton is "in flight" during the transition state. The inhibition patterns by the products are consistent with a random kinetic mechanism.     

Mechanistic requirements for the efficient enzyme-catalyzed hydrolysis of thiosialosides

The sialidase from Micromonospora viridifaciens has been found to catalyze the hydrolysis of aryl 2-thio-alpha-D-sialosides with remarkable efficiency: the first- and second-order rate constants, kcat and kcat/Km, for the enzyme-catalyzed hydrolysis of PNP-S-NeuAc are 196 +/- 5 s(-1) and (6.7 +/- 0.7) x 10(5) M(-1) s(-1), respectively. A reagent panel of eight aryl 2-thio-alpha-D-sialosides was synthesized and used to probe the mechanism for the M. viridifaciens sialidase-catalyzed hydrolysis reaction. In the case of the wild-type enzyme, the derived Br?nsted parameters (beta(lg)) on kcat and kcat/Km are -0.83 +/- 0.11 and -1.27 +/- 0.17 for substrates with thiophenoxide leaving groups of pKa values > or = 4.5. For the general-acid mutant, D92G, the derived beta(lg) value on kcat for the same set of leaving groups is -0.82 +/- 0.12. When the conjugate acid of the departing thiophenol was < or = 4.5, the derived Br?nsted slopes for both the wild-type and the D92G mutant sialidase were close to zero. In contrast, the nucleophilic mutant, Y370G, did not display a similar break in the Br?nsted plots, and the corresponding values for beta(lg), for the three most reactive aryl 2-thiosialosides, on kcat and kcat/Km are -0.76 +/- 0.28 and -0.84 +/- 0.04, respectively. Thus, for the Y370G enzyme glycosidic C-S bond cleavage is rate-determining for both kcat and kcat/Km, whereas, for both the wild-type and D92G mutant enzymes, the presented data are consistent with a change in rate-determining step from glycosidic C-S bond cleavage for substrates in which the pKa of the conjugate acid of the leaving group is > or = 4.5, to either deglycosylation (kcat) or a conformational change that occurs prior to C-S bond cleavage (kcat/Km) for the most activated leaving groups. Thus, the enzyme-catalyzed hydrolysis of 2-thiosialosides is strongly catalyzed by the nucleophilic tyrosine residue, yet the C-S bond cleavage does not require the conserved aspartic acid residue (D92) to act as a general-acid catalyst.     

Experimental charge measurement at leaving oxygen in the bovine ribonuclease A catalyzed cyclization of uridine 3'-phosphate aryl esters

The title esters are demonstrated to be specific substrates of bovine pancreatic ribonuclease A (EC 3.1.27.5). The Br?nsted dependence of kcat/Km at pH 7.50 for the enzyme-catalyzed cyclization versus the pKa of the leaving phenol exhibits two regression lines of almost identical slope for respectively 2-chlorophenols and 2,6-unsubstituted phenols: log kcat/Km = -0.20 pKa ArOH + 5.47 (n = 5, r = 0.957); log kcat/Km = -0.17 pKa ArOH + 5.79 (n = 4, r = 0.965). Comparison of the Br?nsted beta 1g's with that for the standard reaction where imidazole catalyzes the cyclization (beta 1g = -0.59) indicates considerably less development of negative charge on the leaving oxygen in the enzyme case, providing experimental evidence for the hypothesis that electrophilic assistance is involved in catalysis. The existence of two essentially parallel Br?nsted correlations is not reflected in the standard reaction of substrate with imidazole. Modeling studies indicate that the phenyl ring of the substrate can take up a range of positions away from the active site; the presence of ortho chloro substituents considerably restricts the motion of the phenyl leaving group.     

Beta-glucosidase: substrate, solvent, and viscosity variation as probes of the rate-limiting steps

The second-order rate constants (kcat/Km) for the beta-glucosidase-catalyzed hydrolysis of aryl beta-D-glucopyranosides show a bell-shaped dependence of pH. The pKas that characterize this dependence are 4.4 (delta Hion approximately equal to 0) and 6.7 (delta Hion approximately equal to 0). In D2O these pKas are increased by 0.5 (+/- 0.1) unit, but there is no solvent isotope effect on the pH-independent second-order rate constant. Nath and Rydon [Nath, R. L., & Rydon, H. N. (1954) Biochem. J. 57, 1-10] examined the kinetics of the beta-glucosidase-catalyzed hydrolysis of a series of substituted phenyl glucosides. We have extended this study to include glucosides with phenol leaving groups of pKa less than 7. Br?nsted plots for this extended series were nonlinear for both kcat/Km and kcat. Br?nsted coefficients for those compounds with leaving groups of pKa greater than 7 (for kcat/Km) or pKa greater than 8.5 (for kcat) were nearly equal to -1.0, indicating substantial negative charge buildup on the leaving group in the transition state. The nonlinearity indicates an intermediate in the reaction. This was confirmed by partitioning experiments in the presence of methanol as a competing glucose acceptor. A constant product ratio, [methyl glucoside]/[glucose], was found with aryl glucoside substrates varying over 16,000-fold in reactivity (V/K), indicative of a common intermediate. Viscosity variation (in sucrose-containing buffers) was used to probe the extent to which the beta-glucosidase reactions are diffusion-controlled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leaving group dependence in the phosphorylation of Escherichia coli alkaline phosphatase by monophosphate esters

Values of kcat and Km have been measured for the Escherichia coli alkaline phosphatase catalyzed hydrolysis of 18 aryl and 12 alkyl monophosphate esters at pH 8.00 and 25 degrees C. A Br?nsted plot of log (kcat/Km) (M-1 s-1) vs. the pK of the leaving hydroxyl group exhibits two regression lines: log (kcat/Km) = -0.19 (+/- 0.02) pKArOH + 8.14 (+/- 0.15) log (kcat/Km) = -0.19 (+/- 0.01) pKROH + 5.89 (+/- 0.17) Alkyl phosphates with aryl or large lipophilic side chains are not correlated by the above equations and occupy positions intermediate between the two lines. The observed change in effective charge on the leaving oxygen of the ester (-0.2) is very small, consistent with substantial electrophilic participation of the enzyme with this atom. Cyclohexylammonium ion is a noncompetitive inhibitor against 4-nitrophenyl phosphate substrate at pH 8.00, and neutral phenol is a competitive inhibitor (Ki = 82.6 mM); these data and the 100-fold larger reactivity of aryl over alkyl esters are consistent with the existence of a lipophilic binding site for the leaving group of the substrate. The absence of a major steric effect in kcat/Km for substituted aryl esters confirms that the leaving group in the enzyme--substrate complex points away from the surface of the enzyme. Arguments are advanced to exclude a dissociative mechanism (involving a metaphosphate ion) for the enzyme-catalyzed substitution at phosphorus.     

Transient state kinetic studies of the MutT-catalyzed nucleoside triphosphate pyrophosphohydrolase reaction

The MutT pyrophosphohydrolase, in the presence of Mg2+, catalyzes the hydrolysis of nucleoside triphosphates by nucleophilic substitution at Pbeta, to yield the nucleotide and PP(i). The best substrate for MutT is the mutagenic 8-oxo-dGTP, on the basis of its Km being 540-fold lower than that of dGTP. Product inhibition studies have led to a proposed uni-bi-iso kinetic mechanism, in which PP(i) dissociates first from the enzyme-product complex (k3), followed by NMP (k4), leaving a product-binding form of the enzyme (F) which converts to the substrate-binding form (E) in a partially rate-limiting step (k5) [Saraswat, V., et al. (2002) Biochemistry 41, 15566-15577]. Single- and multiple-turnover kinetic studies of the hydrolysis of dGTP and 8-oxo-dGTP and global fitting of the data to this mechanism have yielded all of the nine rate constants. Consistent with an "iso" mechanism, single-turnover studies with dGTP and 8-oxo-dGTP hydrolysis showed slow apparent second-order rate constants for substrate binding similar to their kcat/Km values, but well below the diffusion limit (approximately 10(9) M(-1) s(-1)): k(on)app = 7.2 x 10(4) M(-1) s(-1) for dGTP and k(on)app = 2.8 x 10(7) M(-1) s(-1) for 8-oxo-dGTP. These low k(on)app values are fitted by assuming a slow iso step (k5 = 12.1 s(-1)) followed by fast rate constants for substrate binding: k1 = 1.9 x 10(6) M(-1) s(-1) for dGTP and k1 = 0.75 x 10(9) M(-1) s(-1) for 8-oxo-dGTP (the latter near the diffusion limit). With dGTP as the substrate, replacing Mg2+ with Mn2+ does not change k1, consistent with the formation of a second-sphere MutT-M2+-(H2O)-dGTP complex, but slows the iso step (k5) 5.8-fold, and its reverse (k(-5)) 25-fold, suggesting that the iso step involves a change in metal coordination, likely the dissociation of Glu-53 from the enzyme-bound metal so that it can function as the general base. Multiple-turnover studies with dGTP and 8-oxo-dGTP show bursts of product formation, indicating partially rate-limiting steps following the chemical step (k2). With dGTP, the slow steps are the chemical step (k2 = 10.7 s(-1)) and the iso step (k5 = 12.1 s(-1)). With 8-oxo-dGTP, the slow steps are the release of the 8-oxo-dGMP product (k4 = 3.9 s(-1)) and the iso step (k5 = 12.1 s(-1)), while the chemical step is fast (k2 = 32.3 s(-1)). The transient kinetic studies are generally consistent with the steady state kcat and Km values. Comparison of rate constants and free energy diagrams indicate that 8-oxo-dGTP, at low concentrations, is a better substrate than dGTP because it binds to MutT 395-fold faster, dissociates 46-fold slower, and has a 3.0-fold faster chemical step. The true dissociation constants (KD) of the substrates from the E-form of MutT, which can now be obtained from k(-1)/k1, are 3.5 nM for 8-oxo-dGTP and 62 microM for dGTP, indicating that 8-oxo-dGTP binds 1.8 x 10(4)-fold tighter than dGTP, corresponding to a 5.8 kcal/mol lower free energy of binding.     

Substrate specificity of human fibroblast stromelysin. Hydrolysis of substance P and its analogues

To probe the substrate specificity of the human metalloproteinase stromelysin (SLN), we determined values of kc/Km for the SLN-catalyzed hydrolysis of substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH2; SP; kc/Km = 1790 +/- 140 M-1 s-1), 15 analogues of SP, and 17 other peptides. We found a remarkably narrow substrate specificity for SLN: while SP and its analogues could serve as substrates for SLN (hydrolysis occurred exclusively at the Gln6-Phe7 bond), peptides that were not direct analogues could not (kc/Km less than 3 M-1 s-1). From the study of the SLN-catalyzed hydrolysis of SP and its analogues, the following findings emerged: (1) Decreasing the length of SP results in decreases in kc/Km. (2) Conservative amino acid replacements near the scissle bond of SP decrease kc/Km. (3) The SP analogue in which Gly9 is replaced with sarcosine (N-methylglycine) is not hydrolyzed by SLN (kc/Km less than 3 M-1 s-1). (4) Several SP analogues that are not hydrolyzed by SLN are inhibitors of the enzyme. The complexes formed from interaction of SLN with these peptides have dissociation constants that are similar to the Km value for the complex of SLN and SP. Combined, these results suggest that SLN uses the energy that is available from favorable interactions with its substrate to stabilize catalytic transition states but not the Michaelis complex or other stable-state complexes.     

Identification of the catalytic residues in family 52 glycoside hydrolase,a beta-xylosidase from Geobacillus stearothermophilus T-6

beta-d-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units. The enzymatic hydrolysis of the glycosidic bond involves two carboxylic acid residues, and their identification, together with the stereochemistry of the reaction, provides crucial information on the catalytic mechanism. Two catalytic mutants of a beta-xylosidase from Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to verify their role in catalysis. The activity of the E335G mutant decreased approximately 106-fold, and this activity was enhanced 103-fold in the presence of external nucleophiles such as formate and azide, resulting in a xylosyl-azide product with an opposite anomeric configuration. These results are consistent with Glu335 as the nucleophile in this retaining enzyme. The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pKa). The mutant exhibited 103-fold reduction in activity, and the Br?nsted plot of log(kcat) versus pKa revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 103-fold. The rates of the glycosylation step, as reflected by the specificity constant (kcat/Km), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pKa) but decreased 102-fold for those that require strong acid catalysis (high pKa). Furthermore, the pH dependence profile of the mutant enzyme revealed that acid catalysis is absent. Finally, the presence of azide significantly enhanced the mutant activity accompanied with the generation of a xylosyl-azide product with retained anomeric configuration. These results are consistent with Asp495 acting as the acid-base in XynB2.     

Biochemical and kinetic analysis of the GH3 family beta-xylosidase from Aspergillus awamori X-100

The beta-xylosidase from Aspergillus awamori X-100 belonging to the family 3 glycoside hydrolase revealed a distinctive transglycosylating ability to produce xylooligosaccharides with degree of polymerization more than 7. In order to explain this fact, the enzyme has been subjected to the detailed biochemical study. The enzymatic hydrolysis of p-nitrophenyl beta-D-xylopyranoside was found to occur with overall retention of substrate anomeric configuration suggesting cleavage of xylosidic bonds through a double-displacement mechanism. Kinetic study with aryl beta-xylopyranosides substrates, in which leaving group pK(a)s were in the range of 3.96-10.32, revealed monotonic function of log(k(cat)) and no correlation of log(k(cat)/Km) versus pKa values indicating deglycosylation as a rate-limiting step for the enzymatic hydrolysis. The classical bell-shaped pH dependence of k(cat)/Km indicated two ionizable groups in the beta-xylosidase active site with apparent pKa values of 2.2 and 6.4. The kinetic parameters of hydrolysis, Km and k(cat), of p-nitrophenyl beta-D-1,4-xylooligosaccharides were very close to those for hydrolysis of p-nitrophenyl-beta-D-xylopyranoside. Increase of p-nitrophenyl-beta-D-xylopyranoside concentration up to 80 mM led to increasing of the reaction velocity resulting in k(cat)(app)=81 s(-1). Addition of alpha-methyl D-xylopyranoside to the reaction mixture at high concentration of p-nitrophenyl-beta-D-xylopyranoside (50 mM) caused an acceleration of the beta-xylosidase-catalyzed reactions and appearance of a new transglycosylation product, alpha-methyl D-xylopyranosyl-1,4-beta-D-xylopyranoside, that was identified by 1H NMR spectroscopy. The kinetic model suggested for the enzymatic reaction was consistent with the results obtained.     

Mutagenesis of the conserved active-site tyrosine changes a retaining sialidase into an inverting sialidase

Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase changes the mechanism of catalysis from retention of anomeric configuration to an unprecedented inverting mechanism in which water efficiently functions as the nucleophile. Three mutants, Y370A, Y370D, and Y370G, were produced recombinantly in Escherichia coli, and all are catalytically active against the activated substrate 4-methylumbelliferyl alpha-D-N-acetylneuraminide. The Y370D mutant was also shown to catalyze the hydrolysis of natural substrate analogues such as 3'-sialyllactose. A comparison of the pH-rate profiles for the wild-type and the Y370D mutant sialidase reveals no major differences, although with respect to the kinetic term k(cat)/K(m), an ionized form of the aspartate-370 enzyme is catalytically compromised. For the wild-type enzyme, the value of the Br?nsted parameter beta(lg) on k(cat) is 0.02 +/- 0.03, while for the Y370D mutant sialidase beta(lg) = -0.55 +/- 0.03 for the substrates with bad leaving groups. Thus, for the wild-type enzyme, a nonchemical step(s) is rate-limiting, but for the tyrosine mutant cleavage of the glycosidic C-O bond is rate-determining. The Br?nsted slopes derived for the kinetic parameter k(cat)/K(m) display a similar trend (beta(lg) -0.30 +/- 0.04 and -0.74 +/- 0.04 for the wild-type and Y370D, respectively). These results reveal that the tyrosine residue lowers the activation free energy for cleavage of 6'-sialyllactose, a natural substrate analogue, by more than 24.9 kJ mol(-1). Evidence is presented that the mutant sialidases operate by a dissociative mechanism, and the wild-type enzyme operates by a concerted mechanism.     

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.     

Do enzymes change the nature of transition states? Mapping the transition state for general acid-base catalysis of a serine protease

The properties of the transition state for serine protease-catalyzed hydrolysis of an amide bond were determined for a series of subtilisin variants from Bacillus lentus. There is no significant change in the structure of the enzyme upon introduction of charged mutations S156E/S166D, suggesting that changes in catalytic activity reflect global properties of the enzyme. The effect of charged mutations on the pK(a) of the active site histidine-64 N(epsilon)(2)-H was correlated with changes in the second-order rate constant k(cat)/K(m) for hydrolysis of tetrapeptide anilides at low ionic strength with a Br?nsted slope alpha = 1.1. The solvent isotope effect (D)2(O)(k(cat)/K(m))(1) = 1.4 +/- 0.2. These results are consistent with a rate-limiting breakdown of the tetrahedral intermediate in the acylation step with hydrogen bond stabilization of the departing amine leaving group. There is an increase in the ratio of hydrolysis of succinyl-Ala-Ala-Pro-Phe-anilides for p-nitroaniline versus aniline leaving groups with variants with more basic active site histidines that can be described by the interaction coefficient p(xy) = delta beta(lg)/delta pK(a) (H64) = 0.15. This is attributed to increased hydrogen bonding of the active site imidazolium N-H to the more basic amine leaving group as well as electrostatic destabilization of the transition state. A qualitative characterization of the transition state is presented in terms of a reaction coordinate diagram that is defined by the structure-reactivity parameters.     

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.     

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

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

Dependence of the activity of beef heart mitochondrial adenosinetriphosphatase on the properties of the catalytic metal ion

Several divalent metal ions were used as kinetic probes of the beef heart mitochondrial adenosinetriphosphatase (F1) under a variety of conditions, and the relationship between the properties of the catalytic metal ion and the catalytic activity of the enzyme was examined. Vmax for ATP hydrolysis was largest when metal ions characterized by intermediate values of acidity of coordinated water molecules (pKa) and metal-nucleotide stability constants (Kstab) were present. As temperature increased, the peak of Vmax vs. pKa (or Kstab) shifted to lower initial values of pKa or Kstab. The solvent deuterium isotope effect on Vmax (DV) was normal and largest when the metal ion present during F1-catalyzed ATP hydrolysis was most acidic and the metal nucleotide stability constant was large. When an active site tyrosine on F1 was nitrated, Vmax was most affected when the metal ion present was least acidic and the metal nucleotide stability constant was small. The isotope effect on V/K (DV/K) was normal, small, and apparently independent of the metal ion present. ADP inhibition of F1-catalyzed ATP hydrolysis is competitive, and the Ki is independent of the metal ion present. The degree of Pi inhibition of F1 is dependent on the metal ion present. The inhibition by Pi is competitive at low temperature and becomes noncompetitive as temperature increases. These and previous results support a mechanism whereby a water molecule coordinated to the metal ion of an enzyme-bound gamma-monodentate metal-ATP complex is deprotonated to begin a series of events whereby a beta,gamma-bidentate metal-ATP complex is produced. Upon hydrolysis, the bond between the metal ion and the beta-phosphate of ADP in the Pi-metal-ADP complex is broken before products (ADP and metal-Pi) are released.     

Kinetic analysis of human serine/threonine protein phosphatase 2Calpha.

The PPM family of Ser/Thr protein phosphatases have recently been shown to down-regulate the stress response pathways in eukaryotes. Within the stress pathway, key signaling kinases, which are activated by protein phosphorylation, have been proposed as the in vivo substrates of PP2C, the prototypical member of the PPM family. Although it is known that these phosphatases require metal cations for activity, the molecular details of these important reactions have not been established. Therefore, here we report a detailed biochemical study to elucidate the kinetic and chemical mechanism of PP2Calpha. Steady-state kinetic and product inhibition studies revealed that PP2Calpha employs an ordered sequential mechanism, where the metal cations bind before phosphorylated substrate, and phosphate is the last product to be released. The metal-dependent activity of PP2C (as reflected in kcat and kcat/Km), indicated that Fe2+ was 1000-fold better than Mg2+. The pH rate profiles revealed two ionizations critical for catalytic activity. An enzyme ionization with a pKa value of 7 must be unprotonated for catalysis, and an enzyme ionization with a pKa of 9 must be protonated for substrate binding. Br?nsted analysis of substrate leaving group pKa indicated that phosphomonoester hydrolysis is rate-limiting at pH 7. 0, but not at pH 8.5 where a common step independent of the nature of the substrate and alcohol product limits turnover (kcat). Rapid reaction kinetics between phosphomonoester and PP2C yielded exponential "bursts" of product formation, consistent with phosphate release being the slow catalytic step at pH 8.5. Dephosphorylation of synthetic phosphopeptides corresponding to several protein kinases revealed that PP2C displays a strong preference for diphosphorylated peptides in which the phosphorylated residues are in close proximity.     

Mechanism of Thermoanaerobacterium saccharolyticum beta-xylosidase: kinetic studies

The catalytic mechanism of Thermoanaerobacterium saccharolyticum beta-xylosidase (XynB) from family 39 of glycoside hydrolases has been subjected to a detailed kinetic investigation using a range of substrates. The enzyme exhibits a bell-shaped pH dependence of k(cat)/K(m), reflecting apparent pK(a) values of 4.1 and 6.8. The k(cat) and k(cat)/K(m) values for a series of aryl xylosides have been measured and used to construct two Br?nsted plots. The plot of log(k(cat)/K(m)) against the pK(a) of the leaving group reveals a significant correlation (beta(lg) = -0.97, r(2) = 0.94, n = 8), indicating that fission of the glycosidic bond is significantly advanced in the transition state leading to the formation of the xylosyl-enzyme intermediate. The large negative value of the slope indicates that there is relatively little proton donation to the glycosidic oxygen in the transition state. A biphasic, concave-downward plot of log(k(cat)) against pK(a) provides good evidence for a two-step double-displacement mechanism involving a glycosyl-enzyme intermediate. For activated leaving groups (pK(a) < 9), the breakdown of the xylosyl-enzyme intermediate is the rate-determining step, as indicated by the absence of any effect of the pK(a) of the leaving group on log(k(cat)) (beta(lg) approximately 0). However, a strong dependence of the first-order rate constant on the pK(a) value of relatively poor leaving groups (pK(a) > 9) suggests that the xylosylation step is rate-determining for these substrates. Support for the dexylosylation chemical step being rate-determining for activated substrates comes from nucleophilic competition experiments in which addition of dithiothreitol results in an increase in turnover rates. Normal secondary alpha-deuterium kinetic isotope effects ((alpha-D)(V) or (alpha-D)(V/K) = 1.08-1.10) for three different substrates of widely varying pK(a) value (5.15-9.95) have been measured and these reveal that the transition states leading to the formation and breakdown of the intermediate are similar and both steps involve rehybridization of C1 from sp(3) to sp(2). These results are consistent only with "exploded" transition states, in which the saccharide moiety bears considerable positive charge, and the intermediate is a covalent acylal-ester where C1 is sp(3) hybridized.     

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.     

Pre-steady-state and steady-state kinetic analysis of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.

The complete time course of the hydrolysis of p-nitrophenyl phosphate catalyzed by the low molecular weight (acid) phosphotyrosyl protein phosphatase from bovine heart was elucidated and analyzed in detail. Burst titration kinetics were demonstrated for the first time with this class of enzyme. At pH 7.0, 4.5 degrees C, a transient pre-steady-state "burst" of p-nitrophenol was formed with a rate constant of 48 s-1. The burst was effectively stoichiometric and corresponded to a single enzyme active site/molecule. The burst was followed by a slow steady-state turnover of the phosphoenzyme intermediate with a rate constant of 1.2 s-1. Product inhibition studies indicated an ordered uni-bi kinetic scheme for the hydrolysis. Partition experiments conducted for several substrates revealed a constant product ratio. Vmax was constant for these substrates, and the overall rate of hydrolysis was increased greatly in the presence of alcohol acceptors. An enzyme-catalyzed 18O exchange between inorganic phosphate and water was detected and occurred with kcat = 4.47 x 10(-3) s-1 at pH 5.0, 37 degrees C. These results were all consistent with the existence of a phosphoenzyme intermediate in the catalytic pathway and with the breakdown of the intermediate being the rate-limiting step. The true Michaelis binding constant Ks = 6.0 mM, the apparent Km = 0.38 mM, and the rate constants for phosphorylation (k2 = 540 s-1) and dephosphorylation (k3 = 36.5 s-1) were determined under steady-state conditions with p-nitrophenyl phosphate at pH 5.0 and 37 degrees C in the presence of phosphate acceptors. The energies of activation for the enzyme-catalyzed hydrolysis at pH 5.0 and 7.0 were 13.6 and 14.1 kcal/mol, respectively. The activation energy for the enzyme-catalyzed medium 18O exchange between phosphate and water was 20.2 kcal/mol. Using the available equilibrium and rate constants, an energetic diagram was constructed for the enzyme-catalyzed reaction.     

General base catalysis in a glutamine for histidine mutant at position 51 of human liver alcohol dehydrogenase

On the basis of the three-dimensional structure of horse liver alcohol dehydrogenase determined by X-ray crystallography, His 51 has been proposed to act as a general base during catalysis by abstracting a proton from the alcohol substrate. A hydrogen-bonding system (proton relay system) connecting the alcohol substrate and His 51 has been proposed to mediate proton transfer. We have mutated His 51 to Gln in the homologous human liver beta 1 beta 1 alcohol dehydrogenase isoenzyme which is expected to have a similar proton relay system. The mutation resulted in an about 6-fold drop in V/Kb (Vmax for ethanol oxidation divided by Km for ethanol) at pH 7.0 and a 12-fold drop at pH 6.5. V/Kb could be restored completely or partially by the presence of high concentrations of glycylglycine, glycine, and phosphate buffers. A Br?nsted plot of the effect on V/Kb versus the pKa of these bases plus H2O and OH- was linear. Only secondary or tertiary amine buffers differed from linearity, presumably due to steric hindrance. These results suggest that His 51 acts as a general base catalyst during alcohol oxidation in the wild-type enzyme and can be functionally replaced in the mutant enzyme by general base catalysts present in the solvent. Steady-state kinetic constants for NAD+ and the trifluoroethanol inhibition patterns were similar between the wild-type and the mutant enzyme. Differences in the inhibition constants (Ki) of caprate and trifluoroethanol below pH 7.8 and in the pH dependence of Ki can be explained by the substitution of neutral Gln for positively charged His.