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.     

Sheep kidney pyruvate carboxylase. Studies on the coupling of adenosine triphosphate hydrolysis and CO2 fixation.

Initial velocity and isotope exchange studies confirmed that the over-all reaction, like that catalyzed by pyruvate carboxylase purified from rat liver and chicken liver, was a nonclassical Ping Pong Bi Bi Uni Uni sequence with ATP and HCO3-binding randomly in the Bi Bi partial reaction. Three possible mechanisms for the coupling of ATP hydrolysis and CO2 fixation are considered: (i) Mechanism i, a concerted mechanism without the formation of a kinetically significant or detectable intermediate; (ii) Mechanism ii, activation of the enzyme by ATP to form an activated phosphoenzyme complex which can react with HCO3- by formation of a phosphorylated intermediate. On the basis of other evidence, an activated intermediate containing the ADP moiety was considered improbable. Evidence is presented which indicates that an isotopic exchange between ATP and ADP in the absence of added orthophosphate is not a property of the sheep kidney enzyme. This observation removed the need to postulate either that this exchange is an abortive reaction, or that there is a compulsory formation of a phosphoenzyme intermediate. Two analogues of ADP, alpha,beta-methylene adenosine diphosphate, and adenosine 5'-phosphosulfate, have been used to provide further evidence against Mechanism ii. Both compounds were competitive inhibitors with respect to MgATP2- (Ki values respectively, 0.58 mM and 3.0 mM, compared with 0.17 mM for ADP), but neither could be phosphorylated by the enzyme. Neither analogue could replace ADP in the HCO3-: oxalacetate isotopic exchange reaction, indicating that phosphorylation of ADP is necessary for this exchange to occur, and that Mechanism ii is not applicable. Since Mechanism iii involves formation of a carbonly phosphate intermediate, analogues of this compound, namely, carbamyl phosphate and phosphonacetic acid were used to examine this pathway. The fact that the enzyme catalyzed the synthesis of ATP from ADP and carbamyl phosphate, and that phosphonacetic acid was a noncompetitive inhibitor with respect to MgATP2- (Ki = 0.5 mM) provides strong evidence that a carbonyl phosphate derivative is involved in the reaction mechanism. However, we have not found from initial velocity studies evidence for the formation of this intermediate, and it may therefore have only a transient existence in an essentially concerted reaction.     

Transition-state analysis of a Vmax mutant of AMP nucleosidase by the application of heavy-atom kinetic isotope effects

The transition state of the Vmax mutant of AMP nucleosidase from Azotobacter vinelandii [Leung, H. B., & Schramm, V. L. (1981) J. Biol. Chem. 256, 12823-12829] has been characterized by heavy-atom kinetic isotope effects in the presence and absence of MgATP, the allosteric activator. The enzyme catalyzes hydrolysis of the N-glycosidic bond of AMP at approximately 2% of the rate of the normal enzyme with only minor changes in the Km for substrate, the activation constant for MgATP, and the Ki for formycin 5'-phosphate, a tight-binding competitive inhibitor. Isotope effects were measured as a function of the allosteric activator concentration that increases the turnover number of the enzyme from 0.006 s-1 to 1.2 s-1. The kinetic isotope effects were measured with the substrates [1'-3H]AMP, [2'-2H]AMP, [2'-2H]AMP, [9-15N]AMP, and [1',9-14C, 15N]AMP. All substrates gave significant kinetic isotope effects in a pattern that establishes that the reaction expresses intrinsic kinetic isotope effects in the presence or absence of MgATP. The kinetic isotope effect with [9-15N]AMP decreased from 1.034 +/- 0.002 to 1.021 +/- 0.002 in response to MgATP. The [1'-3H]AMP isotope effect increased from 1.086 +/- 0.003 to 1.094 +/- 0.002, while the kinetic isotope effect for [1',9-14C, 15N]AMP decreased from 1.085 +/- 0.003 to 1.070 +/- 0.004 in response to allosteric activation with MgATP. Kinetic isotope effects with [1'-14C]AMP and [2'-2H]AMP were 1.041 +/- 0.006 and 1.089 +/- 0.002 and were not changed by addition of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbamoyl-phosphate synthetase II of the mammalian CAD protein: kinetic mechanism and elucidation of reaction intermediates by positional isotope exchange

The kinetic mechanism of carbamoyl-phosphate synthetase II from Syrian hamster kidney cells has been determined at pH 7.2 and 37 degrees C. Initial velocity, product inhibition, and dead-end inhibition studies of both the biosynthetic and bicarbonate-dependent adenosinetriphosphatase (ATPase) reactions are consistent with a partially random sequential mechanism in which the ordered addition of MgATP, HCO3-, and glutamine is followed by the ordered release of glutamate and Pi. Subsequently, the binding of a second MgATP is followed by the release of MgADP, which precedes the random release of carbamoyl phosphate and a second MgADP. Carbamoyl-phosphate synthetase II catalyzes beta gamma-bridge:beta-nonbridge positional oxygen exchange of [gamma-18O]ATP in both the ATPase and biosynthetic reactions. Negligible exchange is observed in the strict absence of HCO3- (and glutamine or NH4+). The ratio of moles of MgATP exchanged to moles of MgATP hydrolyzed (nu ex/nu cat) is 0.62 for the ATPase reaction, and it is 0.39 and 0.16 for the biosynthetic reaction in the presence of high levels of glutamine and NH4+, respectively. The observed positional isotope exchange is suppressed but not eliminated at nearly saturating concentrations of either glutamine or NH4+, suggesting that this residual exchange results from either the facile reversal of an E-MgADP-carboxyphosphate-Gln(NH4+) complex or exchange within an E-MgADP-carbamoyl phosphate-MgADP complex, or both. In the 31P NMR spectra of the exchanged [gamma-18O]ATP, the distribution patterns of 16O in the gamma-phosphorus resonances in all samples reflect an exchange mechanism in which a rotationally unhindered molecule of [18O3, 16O]Pi does not readily participate. These results suggest that the formation of carbamate from MgATP, HCO3-, and glutamine proceeds via a stepwise, not concerted mechanism, involving at least one kinetically competent covalent intermediate, such as carboxyphosphate.     

Permeable membrane/mass spectrometric measurement of solvent 1H/2H, 12C/13C, and 16O/18O kinetic isotope effects associated with alpha-chymotrypsin deacylation: evidence for reaction mechanism plasticity

We have measured, by permeable membrane/mass spectrometry, the 16O/18O, 12C/13C, and solvent H2O/D2O kinetic isotope effects (kie) associated with acyl-alpha-chymotrypsin hydrolysis and transesterification. The hydrolysis of alpha-chymotrypsinyl 2-furoate has a 12C/13C kie of approximately 1.06. Transesterification of the same acyl enzyme shows 16O/18O, 12C/13C, and solvent H2O/D2O kinetic isotope effects of 1.015 (0.003), 1.01-1.02, and 2.226 (0.007), respectively. From the temperature independence of the 16O/18O transesterification kinetic isotope effect and kinetic data reported elsewhere [Wang, C.-L. A., Calvo, K. C., & Klapper, M. H. (1981) Biochemistry 20, 1401-1408], we conclude that there are two active forms of acylchymotrypsin. We also propose that formation of the tetrahedral intermediate is the rate-limiting step in both hydrolysis and transesterification and that the position of the transition state in the transesterification is closer to the starting enzyme ester while that for the hydrolytic reaction is closer to the tetrahedral intermediate. These results are discussed in terms of reaction mechanism plasticity.     

Magnesium-dependent ATPase activity and cooperativity of magnesium chelatase from Synechocystis sp. PCC6803

The first committed step in chlorophyll biosynthesis is catalyzed by magnesium chelatase, a complex enzyme with at least three substrates, cooperative Mg(2+) activation, and free energy coupling between ATP hydrolysis and metal-ion chelation. A detailed functional study of the behavior of the intact magnesium chelatase has been performed, including characterization of magnesium cooperativity and the stoichiometry of ATP consumption in relation to the magnesium porphyrin produced. It is demonstrated that, in vitro, this catalyzed reaction requires hydrolysis of approximately 15 MgATP(2-) and that the chelation partial reaction is energetically unfavorable, under our assay conditions, with a DeltaG degrees ' of 25-33 kJ mol(-1). Given the likely metabolite concentrations in vivo, this results in the chelatase reaction operating far from equilibrium. We have also determined the steady-state kinetic behavior of the intact enzyme and have compared the kinetic parameters obtained with those observed for the partial reactions of individual subunits. K(DIX) (where D(IX) represents deuteroporphyrin IX) is estimated to be 3.20 microm, and K(MgATP)(2-) is 0.45 mm. k(cat) for chelation is estimated to be 0.8 min(-1), suggesting that the ATP hydrolysis catalyzed by the isolated ChlI subunit is substantially slower in the intact chelatase. The magnesium-rich form of the chelatase is a more effective catalyst of the chelation reaction; magnesium activation of the chelatase increases V, as well as the specificity constant for the reaction of MgATP(2-) and D(IX), possibly as a result of a magnesium-triggered conformational change.     

Steady-state kinetics and isotope effects on the mutant catalytic trimer of aspartate transcarbamoylase containing the replacement of histidine 134 by alanine.

A detailed kinetic analysis of the catalytic trimer of aspartate transcarbamoylase containing the active site substitution H134A was performed to investigate the role of His 134 in the catalytic mechanism. Replacement of histidine by alanine resulted in decreases in the affinities for the two substrates, carbamoyl phosphate and aspartate, and the inhibitor succinate, by factors of 50, 10, and 6, respectively, and yielded a maximum velocity that was 5% that of the wild-type enzyme. However, the pK values determined from the pH dependence of the kinetic parameters, log V and log (V/K) for aspartate, the pK(i) for succinate, and the pK(ia) for carbamoyl phosphate, were similar for both the mutant and the wild-type enzymes, indicating that the protonated form of His 134 does not participate in binding and catalysis between pH 6.2 and 9.2. 13C and 15N isotope effects were studied to determine which steps in the catalytic mechanism were altered by the amino acid substitutions. The 13(V/K) for carbamoyl phosphate exhibited by the catalytic trimer containing alanine at position 134 revealed an isotope effect of 4.1%, probably equal to the intrinsic value and, together with quantitative analysis of the 15N isotope effects, showed that formation of the tetrahedral intermediate is rate-determining for the mutant enzyme. Thus, His 134 plays a role in the chemistry of the reaction in addition to substrate binding. The initial velocity pattern for the reaction catalyzed by the H134A mutant intersected to the left of the vertical axis, negating an equilibrium ordered kinetic mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

The catalytic mechanism of the amidotransferase domain of the Syrian hamster multifunctional protein CAD. Evidence for a CAD-glutamyl covalent intermediate in the formation of carbamyl phosphate.

The multifunctional protein CAD catalyzes the first three steps in pyrimidine biosynthesis in mammalian cells, including the synthesis of carbamyl phosphate from bicarbonate, MgATP and glutamine. The Syrian hamster CAD glutaminase (GLNase) domain, a trpG-type amidotransferase, catalyzes glutamine hydrolysis in the absence of MgATP and bicarbonate (Km = 95 microM and kcat = 0.14 s-1). Unlike E. coli carbamyl phosphate synthetase (Wellner, V.P., Anderson, P.M., and Meister, A. (1973) Biochemistry 12, 2061-2066), a stable thioester intermediate did not accumulate when the mammalian enzyme was incubated with glutamine. However, a covalent adduct could be isolated when the protein was denatured in acid. The steady state concentration of the intermediate increased with increasing glutamine concentration to nearly one mole per mole of enzyme with half saturation at 105 microM, close to the Km value for glutamine. The adduct formed at the active site of the glutaminase domain. The rate of breakdown of the intermediate (k4), determined directly, was 0.17 s-1 and the rate of formation (k3) was estimated as 0.52 s-1. In the absence of MgATP and bicarbonate, k4 = kcat indicating that the decomposition of the intermediate is the rate-limiting step. The intermediate was chemically and kinetically competent, and the glutamine dissociation constant (330 microM) and rate constants were consistent with steady state kinetics and accurately predicted the steady state concentration of the intermediate. These studies suggest a mechanism similar to the cysteine proteases such as recently proposed by Mei and Zalkin (Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264, 16613-16619) who identified a catalytic triad in glutamine phosphoribosyl-5'-pyrophosphate amidotransferase, a purF-type enzyme. MgATP and bicarbonate increased kcat of the glutaminase reaction 14-fold by accelerating both the rate of formation and the rate of breakdown of the intermediate, and prevented the accumulation of the intermediate; however, the Km value for glutamine was not significantly altered. The instability of the thioester intermediate leads to appreciable hydrolysis of glutamine in the absence of the other substrates. However, bicarbonate alone spares glutamine by increasing the Km and Ks of glutamine to 600 and 8960 microM, respectively, thus reducing kcat/Km 3-fold when MgATP is limiting. In the absence of MgATP and bicarbonate, ammonia decreased the rate of hydrolysis and the accumulation of the thioester intermediate indicating that ammonia had direct access to the thioester at the GLNase domain active site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism and activation for allosteric adenosine 5'-monophosphate nucleosidase. Kinetic alpha-deuterium isotope effects for the enzyme-catalyzed hydrolysis of adenosine 5'-monophosphate and nicotinamide mononucleotide

The kinetic alpha-deuterium isotope effect on Vmax/Km for hydrolysis of NMN catalyzed by AMP nucleosidase at saturating concentrations of the allosteric activator MgATP2- is kH/kD = 1.155 +/- 0.012. This value is close to that reported previously for the nonenzymatic hydrolysis of nucleosides of related structure, suggesting that the full intrinsic isotope effect for enzymatic NMN hydrolysis is expressed under these conditions; that is, bond-changing reactions are largely or completely rate-determining and the transition state has marked oxocarbonium ion character. The kinetic alpha-deuterium isotope effect for this reaction is unchanged when deuterium oxide replaces water as solvent, corroborating this conclusion. Furthermore, this isotope effect is independent of pH over the range 6.95-9.25, for which values of Vmax/Km change by a factor of 90, suggesting that the isotope-sensitive and pH-sensitive steps for AMP-nucleosidase-catalyzed NMN hydrolysis are the same. Values of kH/kD for AMP nucleosidase-catalyzed hydrolysis of NMN decrease with decreasing saturation of enzyme with MgATP2- and reach unity when the enzyme is less than half-saturated with this activator. This requires that the rate-determining step changes from cleavage of the covalent C-N bond to one which is isotope-independent. In contrast to the case for NMN hydrolysis, AMP nucleosidase-catalyzed hydrolysis of AMP at saturating concentrations of MgATP2- shows a kinetic alpha-deuterium isotope effect of unity. Thus, covalent bond-changing reactions are largely or completely rate-determining for hydrolysis of a poor substrate, NMN, but make little or no contribution to rate-determining step for hydrolysis of a good substrate, AMP, by maximally activated enzyme. This behavior has several precedents.     

Role of protein conformational mobility in enzyme catalysis: acylation of alpha-chymotrypsin by specific peptide substrates

To probe the mechanistic origins of convex Eyring plots that have been observed for alpha-chymotrypsin (alpha-CT)-catalyzed hydrolysis of specific p-nitroanilide substrates [Case, A., and Stein, R. L. (2003) Biochemistry 42, 3335-3348], we determined the temperature-dependence of (15)N-kinetic isotope effects for the alpha-CT-catalyzed hydrolysis of N-succinyl-Phe p-nitroanilide (Suc-Phe-pNA). To provide an interpretational context for these enzymatic isotope effects, we also determined 15N-KIE for alkaline hydrolysis of p-nitroacetanilide. In 0.002 and 2 N hydroxide (30 degrees C), 15N-KIE values are 1.035 and 0.995 (+/-0.001), respectively, and are consistent with the reported [HO-]-dependent change in rate-limiting step from leaving group departure from an anionic tetrahedral intermediate in dilute base, to hydroxide attack in concentrated base. For the alpha-CT-catalyzed hydrolysis of Suc-Phe-pNA, 15N-KIE is on kc/Km and thus reflects structural features of transition states for all reaction steps up to and including acylation of the active site serine. The isotope effect at 35 degrees C is 1.014 (+/-0.001) and suggests that in the transition state for this reaction, departure of leaving group from the tetrahedral intermediate is well advanced. Significantly, 15N-KIE does not vary over the temperature range 5-45 degrees C. This result eliminates one of the competing hypotheses for the convex Eyring plot observed for this reaction, that is, a temperature-dependent change in rate-limiting step within the chemical manifold of acylation, but supports a mechanism in which an isomerization of enzyme conformation is coupled to active site chemistry. We finally suggest that the near absolute temperature-independence of 15N-KIE may point to a unique transition state for this process.     

Positional isotope exchange and kinetic experiments with Escherichia coli guanosine-5'-monophosphate synthetase

The kinetic mechanism of Escherichia coli guanosine-5'-monophosphate synthetase has been determined by utilizing initial velocity kinetic patterns and positional isotope exchange experiments. The initial velocity patterns of MgATP, XMP, and either NH3 or glutamine (as nitrogen source) were consistent with the ordered addition of MgATP followed by XMP and then NH3. The enzyme catalyzes the exchange of 18O from the beta-nonbridge positions of [beta,beta,beta gamma,gamma,gamma,gamma-18O6]ATP into the alpha beta-bridge position only in the presence of XMP and Mg2+. The exchange reaction did not require NH3. The isotope exchange reaction increased as the XMP concentration increased and then decreased at saturating levels of XMP. These results also support the ordered addition of MgATP followed by XMP. GMP synthetase catalyzes the hydrolysis of ATP to AMP and PPi along with an ATP/PPi exchange reaction in the absence of NH3. These data taken together support a mechanism in which the initial step in the enzymatic reaction involves formation of an adenyl-XMP intermediate. Psicofuranine, an irreversible inhibitor of the enzyme, acts by preventing the release or further reaction of adenyl-XMP with H2O or NH3 but does not suppress the isotope exchange or ATP/PPi exchange reactions. GMP synthetase has also been shown to require a free divalent cation for full activity. When Ca2+ replaces Mg2+ in the reaction, the positional isotope exchange reaction is enhanced but the reaction with NH3 to form GMP is greatly suppressed.     

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.     

Transition state analysis and requirement of Asp-262 general acid/base catalyst for full activation of dual-specificity phosphatase MKP3 by extracellular regulated kinase

Dual-specificity phosphatase MKP3 down-regulates mitogenic signaling through dephosphorylation of extracellular regulated kinase (ERK). Unlike a simple substrate-enzyme interaction, the noncatalytic, amino-terminal domain of MKP3 can bind efficiently to ERK, leading to activation of the phosphatase catalytic domain by as much as 100-fold toward exogenous substrates. It has been suggested that ERK activates MKP3 through the stabilization of the active phosphatase conformation, enabling general acid catalysis. Here, we investigated whether Asp-262 of MKP3 is the bona fide general acid and evaluated its contribution to the catalytic steps activated by ERK. Using site-directed mutagenesis, pH rate and Br?nsted analyses, kinetic isotope effects, and steady-state and rapid reaction kinetics, Asp-262 was identified as the authentic general acid catalyst, donating a proton to the leaving group oxygen during P-O bond cleavage. Kinetic isotope effects [(18)(V/K)(bridge), (18)(V/K)(nonbridge), and (15)(V/K)] were evaluated for the effect of ERK and of the D262N mutation on the transition state of the phosphoryl transfer reaction. The patterns of the three isotope effects for the reaction with native MKP3 in the presence of ERK are indicative of a reaction where the leaving group is protonated in the transition state, whereas in the D262N mutant, the leaving group departs as the anion. Even without general acid catalysis, the D262N mutant reaction is activated by ERK through increased phosphate affinity ( approximately 8-fold) and the partial stabilization of the transition state for phospho-enzyme intermediate formation ( approximately 4-fold). Based on these analyses, we estimate that dephosphorylation of phosphorylated ERK by the D262N mutant is >1000-fold lower than by native, activated MKP3. Also, the kinetic results suggest that Asp-262 functions as a general base during thiol-phosphate intermediate hydrolysis.     

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)     

Role of the four conserved histidine residues in the amidotransferase domain of carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from ATP, bicarbonate, and glutamine. The amidotransferase activity of this enzyme is catalyzed by the smaller of the two subunits of the heterodimeric protein. The roles of four conserved histidine residues within this subunit were probed by site-directed mutagenesis to asparagine. The catalytic activities of the H272N and H341N mutants are not significantly different than that of the wild-type enzyme. The H353N mutant is unable to utilize glutamine as a nitrogen source in the synthetase reaction or the partial glutaminase reaction. However, binding to the glutamine active site is not impaired in the H353N enzyme since glutamine is found to activate the partial ATPase reaction by 40% with a Kd of 54 microM. The H312N mutant has a Michaelis constant for glutamine that is 2 orders of magnitude larger than the wild-type value, but the maximal rate of glutamine hydrolysis is unchanged. These results are consistent with His-353 functioning as a general acid/base catalyst for proton transfers while His-312 serves a critical role for the binding of glutamine to the active site.     

Catalytic and allosteric mechanism of AMP nucleosidase from primary, beta-secondary, and multiple heavy atom kinetic isotope effects

Adenosine 5'-phosphate was synthesized with specific heavy atom substitutions to permit measurement of V/K kinetic isotope effects for the N-glycohydrolase activity of the allosteric AMP nucleosidase and the acid-catalyzed solvolysis of these compounds. The effects of allosteric activation on the kinetic isotope effects together with the kinetic mechanism of AMP nucleosidase [DeWolf, W. E., Jr., Emig, F. A., & Schramm, V. L. (1986) Biochemistry 25, 4132-4140] indicate that the kinetic isotope effects are fully expressed. Comparison of individual primary and secondary kinetic isotope effects with combined isotope effects and the isotope effect of the reverse reaction indicated that kinetic isotope effects in AMP nucleosidase arise from a single step in the reaction mechanism. Under these conditions, kinetic isotope effects can be used to interpret transition-state structure for AMP nucleosidase. Changes in kinetic isotope effects occurred as a function of allosteric activator, demonstrating that allosteric activation alters transition-state structure for AMP nucleosidase. Kinetic isotope effects, expressed as [V/K(normal isotope]/[V/K(heavy isotope)], were observed with [2'-2H]AMP (1.061 +/- 0.002), [9-15N]AMP (1.030 +/- 0.003), [1'-2H]AMP (1.045 +/- 0.002), and [1'-14C]AMP (1.035 +/- 0.002) when hydrolyzed by AMP nucleosidase in the absence of MgATP. Addition of MgATP altered the [2'-2H]AMP effect (1.043 +/- 0.002) and the [1'-2H]AMP effect (1.030 +/- 0.003) and caused a smaller decrease of the 14C and 15N effects. Multiple heavy atom substitutions into AMP caused an increase in observed isotope effects to 1.084 +/- 0.004 for [1'-2H,1'-14C]AMP and to 1.058 +/- 0.002 for [9-15N,1'-14C]AMP with the enzyme in the absence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional linkage between the glutaminase and synthetase domains of carbamoyl-phosphate synthetase. Role of serine 44 in carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (cad).

Mammalian carbamoyl-phosphate synthetase is part of carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD), a multifunctional protein that also catalyzes the second and third steps of pyrimidine biosynthesis. Carbamoyl phosphate synthesis requires the concerted action of the glutaminase (GLN) and carbamoyl-phosphate synthetase domains of CAD. There is a functional linkage between these domains such that glutamine hydrolysis on the GLN domain does not occur at a significant rate unless ATP and HCO(3)(-), the other substrates needed for carbamoyl phosphate synthesis, bind to the synthetase domain. The GLN domain consists of catalytic and attenuation subdomains. In the separately cloned GLN domain, the catalytic subdomain is down-regulated by interactions with the attenuation domain, a process thought to be part of the functional linkage. Replacement of Ser(44) in the GLN attenuation domain with alanine increases the k(cat)/K(m) for glutamine hydrolysis 680-fold. The formation of a functional hybrid between the mammalian Ser(44) GLN domain and the Escherichia coli carbamoyl-phosphate synthetase large subunit had little effect on glutamine hydrolysis. In contrast, ATP and HCO(3)(-) did not stimulate the glutaminase activity, indicating that the interdomain linkage had been disrupted. In accord with this interpretation, the rate of glutamine hydrolysis and carbamoyl phosphate synthesis were no longer coordinated. Approximately 3 times more glutamine was hydrolyzed by the Ser(44) --> Ala mutant than that needed for carbamoyl phosphate synthesis. Ser(44), the only attenuation subdomain residue that extends into the GLN active site, appears to be an integral component of the regulatory circuit that phases glutamine hydrolysis and carbamoyl phosphate synthesis.     

Uridine phosphorylase from Trypanosoma cruzi: kinetic and chemical mechanisms

The reversible phosphorolysis of uridine to generate uracil and ribose 1-phosphate is catalyzed by uridine phosphorylase and is involved in the pyrimidine salvage pathway. We define the reaction mechanism of uridine phosphorylase from Trypanosoma cruzi by steady-state and pre-steady-state kinetics, pH-rate profiles, kinetic isotope effects from uridine, and solvent deuterium isotope effects. Initial rate and product inhibition patterns suggest a steady-state random kinetic mechanism. Pre-steady-state kinetics indicated no rate-limiting step after formation of the enzyme-products ternary complex, as no burst in product formation is observed. The limiting single-turnover rate constant equals the steady-state turnover number; thus, chemistry is partially or fully rate limiting. Kinetic isotope effects with [1'-(3)H]-, [1'-(14)C]-, and [5'-(14)C,1,3-(15)N(2)]uridine gave experimental values of (α-T)(V/K)(uridine) = 1.063, (14)(V/K)(uridine) = 1.069, and (15,β-15)(V/K)(uridine) = 1.018, in agreement with an A(N)D(N) (S(N)2) mechanism where chemistry contributes significantly to the overall rate-limiting step of the reaction. Density functional theory modeling of the reaction in gas phase supports an A(N)D(N) mechanism. Solvent deuterium kinetic isotope effects were unity, indicating that no kinetically significant proton transfer step is involved at the transition state. In this N-ribosyl transferase, proton transfer to neutralize the leaving group is not part of transition state formation, consistent with an enzyme-stabilized anionic uracil as the leaving group. Kinetic analysis as a function of pH indicates one protonated group essential for catalysis and for substrate binding.     

Slow-binding inhibition of gamma-glutamyl transpeptidase by gamma-boroGlu

gamma-Glutamyl transpeptidase (gammaGTase) catalyzes the transfer of the gamma-glutamyl moiety of gamma-glutamyl-derived peptides, such as glutathione (gammaGlu-Cys-Gly), and anilides, such as gamma-glutamyl-7-amido-4-methylcoumarin (gammaGlu-AMC), to acceptor molecules, including water and various dipeptides. These acyl-transfer reactions all occur through a common acyl-enzyme intermediate formed from attack of an active site hydroxyl on the gamma-carbonyl carbon of gammaGlu-X with displacement of X. In this paper, we report that gammaGTase is potently inhibited by the gamma-boronic acid analogue of L-glutamic acid, 3-amino-3-carboxypropaneboronic acid (gamma-boroGlu). We propose that gamma-boroGlu adds to the active site hydroxyl of gammaGTase to form a covalent, tetrahedral adduct that resembles tetrahedral transition states and intermediates that occur along the reaction pathway for gammaGTase-catalyzed reactions. Our studies demonstrate that gamma-boroGlu is a competitive inhibitor of the gammaGTase-catalyzed hydrolysis of gammaGlu-AMC with a K(i) value of 35 nM. Kinetics of inhibition studies allow us to estimate the following values: k(on) = 400 mM(-1) s(-1) and k(off) = 0.02 s(-1). We also found that gamma-boroGlu is an uncompetitive inhibitor of Gly-Gly-promoted transamidation of gammaGlu-AMC. This observation is consistent with the kinetic mechanism we determined for gammaGTase-catalyzed transamidation of gammaGlu-AMC by Gly-Gly to form gammaGlu-Gly-Gly. To probe rate-limiting transition states for gammaGTase catalysis and inhibition, we determined solvent deuterium isotope effects. Solvent isotope effects on k(c)/K(m) for hydrolysis of gammaGlu-AMC and k(on) for inhibition by gamma-boroGlu are identical and equal unity, suggesting that the processes governed by these rate constants are both rate-limited by a step that is insensitive to solvent deuterium such as a conformational fluctuation of the initially formed E-S or E-I complex. In contrast, the solvent isotope effect on k(c) is 2.4. k(c) is rate-limited by hydrolysis of the acyl-enzyme intermediate that is formed during reaction of gammaGTase with gammaGlu-AMC. Thus, the magnitude of this isotope effect suggests the formation of a catalytically important protonic bridge in the rate-limiting transition state for deacylation.     

Mutagenic studies on histidine 98 of methylglyoxal synthase: effects on mechanism and conformational change

Two detailed mechanisms [Marks et al. (2001) Biochemistry 40, 6805] have been proposed to explain the activity of methylglyoxal synthase (MGS), a homohexameric allosterically regulated enzyme that catalyzes the elimination of phosphate from DHAP to form enol pyruvaldehyde. This enol then tautomerizes to methylglyoxal in solution. In one of these mechanisms His 98 plays an obligate role in the transfer of a proton from the O(3) oxygen of DHAP to the O2 oxygen. To test this hypothesized mechanism, the variants H98N and H98Q were expressed and purified. Relative to the wild-type enzyme, the H98N variant shows a 50-fold decrease in k(cat) with all other kinetic parameters (i.e., K(m), K(PGA), etc.) being nearly the same. By contrast, the apparent catalytic rate for the H98Q variant is 250-fold lower than that of the wild-type enzyme. Inorganic phosphate acts as a competitive inhibitor (with a K(i) of 15 microM) rather than as an allosteric-type inhibitor as it does in the wild-type enzyme, and the competitive inhibitor phosphoglyolate (PGA) acts as an activator of this variant. These facts are explained by a shift in the conformational equilibrium toward an "inactive" state. When activation by PGA is accounted for, the catalytic rate for the "active" state of H98Q is estimated to be only 6-fold less than that of the wild-type enzyme, and thus His 98 is not essential for catalysis. Primary deuterium isotope effect data were measured for the wild-type enzyme and the two variants. At pH 7.0, the (D)V isotope effect (1.5) and the absence of a (D)(V/K) isotope effect for the wild-type enzyme suggest that the rate for the isotopically sensitive step is partially rate limiting but greater than the rate of substrate dissociation. Both the (D)V (2.0) and (D)(V/K) (3.4) isotope effects were more pronounced in the H98N variant, while the H98Q variant displayed an unusual inverse (D)V (0.8) isotope effect and a normal (D)(V/K) (1.5) isotope effect. The X-ray crystal structures of PGA bound to the H98Q and H98N variants were both determined to a resolution of 2.2 A. These mutations had little effect on the overall structure. However, the pattern of hydrogen bonding in the active site suggests an explanation as to how in the wild-type and H98N mutated enzymes the "inactive to active" equilibrium lies toward the active state, while with the H98Q mutated enzyme the equilibrium lies toward the inactive state. Thus, the role of His 98 appears to be more as a regulator of the enzyme's conformation rather than as a critical contributor to the catalytic mechanism.