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M H O'Leary

Publications and source records attributed to M H O'Leary.

At least 19 recordsLinked to original sources
PubMed1998

Carbon-13 isotope effect studies of Trypanosoma brucei ornithine decarboxylase.

Carbon isotope effect studies were undertaken with the wild-type pyridoxal 5'-phosphate (PLP)-dependent enzyme ornithine decarboxylase (ODC) from Trypanosoma brucei and with several active site mutants of the enzyme. For the decarboxylation of the optimal substrate, L-ornithine, by wild-type ODC, the observed carbon isotope effect (k12/k13) is 1.033 at pH 7.3. In comparison to the expected intrinsic isotope effect (k12/k13 = 1.06) for decarboxylation, this value suggests that both the rate of decarboxylation and the rate of Schiff base interchange with L-ornithine are partially rate-limiting for the reaction steps up to decarboxylation. In contrast, with the alternate substrate L-Lys, which shows lower catalytic efficiency, the carbon isotope effect increased to 1.063, demonstrating that decarboxylation has become the rate-limiting step. For the mutant enzymes, E274A ODC and C360A ODC, with L-ornithine as substrate the carbon isotope effect also approaches the intrinsic limit. Glu-274 was previously demonstrated to play a direct role in carbanion stabilization, and thus the large carbon isotope effect (k12/k13 = 1.055) is consistent with an impaired rate of decarboxylation compared to wild-type ODC. In contrast, for K69A ODC, the isotope effect is almost entirely suppressed, suggesting that Schiff-base formation (which now must occur from enzyme-bound PLP, rather than from an enzyme-bound PLP-Schiff base) has become rate-determining.

Animals↗
PubMed1996

Probing the mechanism of nitrogen transfer in Escherichia coli asparagine synthetase by using heavy atom isotope effects.

In experiments aimed at determining the mechanism of nitrogen transfer in purF amidotransferase enzymes, 13C and 15N kinetic isotope effects have been measured for both of the glutamine-dependent activities of Escherichia coli asparagine synthetase B (AS-B). For the glutaminase reaction catalyzed by AS-B at pH 8.0, substitution heavy atom labels in the side chain amide of the substrate yields observed values of 1.0245 and 1.0095 for the amide carbon and amide nitrogen isotope effects, respectively. In the glutamine-dependent synthesis of asparagine at pH 8.0, the amide carbon and amide nitrogen isotope effects have values of 1.0231 and 1.0222, respectively. We interpret these results to mean that nitrogen transfer does not proceed by the formation of free ammonia in the active site of the enzyme and probably involves a series of intermediates in which glutamine becomes covalently attached to aspartate. While a number of mechanisms are consistent with the observed isotope effects, a likely reaction pathway involves reaction of an oxyanion with beta-aspartyl-AMP. This yields an intermediate in which C-N bond cleavage gives an acylthioenzyme and a second tetrahedral intermediate. Loss of AMP from the latter gives asparagine. An alternate reaction mechanism in which asparagine is generated from an imide intermediate also appears consistent with the observed kinetic isotope effects.

Aspartate-Ammonia Ligase↗
PubMed1995

Domain closure in the catalytic chains of Escherichia coli aspartate transcarbamoylase influences the kinetic mechanism.

The closure of the two domains of the catalytic chains of Escherichia coli aspartate transcarbamoylase, which is critical for completion of the T-->R transition, is stabilized by salt-bridges between Glu-50 and both Arg-167 and Arg-234. Mutation of Glu-50 to Ala shifts the enzyme toward a low activity, low affinity state (Newton, C. J., and Kantrowitz, E. R. (1990) Biochemistry, 29, 1444-1451). Kinetic isotope effects (KIE) and equilibrium isotope exchange kinetics (EIEK) have been used to probe the dynamic properties of the Glu-50-->Ala enzyme. Unlike the behavior of the wild-type enzyme, the observed kinetic isotope effect for 13C versus 12C at the carbonyl group of carbamoyl phosphate (CP) increased upon the binding of ligands which promote the formation of the R-state (Asp, N-phosphonacetyl-L-aspartate (PALA), or ATP). The maximum rate for the [14C]Asp<-->Carbamoyl aspartate (CAsp) exchange with the Glu-50-->Ala enzyme was 500-fold slower than for the wild-type enzyme; however, the rate for the [14C]CP<-->CAsp exchange was only 50-fold slower, reversing the relative rates observed with the wild-type enzyme. In addition, upon variation of substrate pairs involving Asp or CAsp, loss of inhibition effects in the CP<-->CAsp exchange indicated that the Glu-50-->Ala substitution caused the kinetic mechanism for the mutant enzyme to shift from ordered to random. Computer simulations of the EIEK data indicate that the Glu-50-->Ala mutation specifically causes strong decreases in the rates of catalysis and association-dissociation for Asp and CAsp, with minimal effects on the CP and Pi on-off rates. With substrates bound, the Glu-50-->Ala enzyme apparently does not attain a full R-state conformation. The PALA-activated Glu-50-->Ala enzyme, however, exhibits substrate affinities comparable to those for the wild-type enzyme, but fails to restore the preferred order substrate binding. Unlike the wild-type enzyme, both the T and R-states of the Glu-50-->Ala enzyme contribute to catalysis. A third state, I, is proposed for the Glu-50-->Ala enzyme, in which random order substrate binding is exhibited, and the catalytic step contributes significantly to overall rate limitation.

Adenosine Triphosphate↗
PubMed1995

L-aspartate association contributes to rate limitation and induction of the T-->R transition in Escherichia coli aspartate transcarbamoylase. Equilibrium exchanges and kinetic isotope effects with a Vmax-enhanced mutant, Asp-236-->Ala.

Equilibrium isotope exchange kinetics (EIEK) and kinetic isotope effects have been used to determine the mechanistic basis for the altered kinetic characteristics of a mutant version of Escherichia coli aspartate transcarbamylase in which Asp-236 of the catalytic chain is replaced by alanine (Asp-236-->Ala). The [14C]Asp<--> N-carbamyl-L-aspartate (CAsp) and [14C]CP<-->CAsp exchange rates, observed as a function of various reactant-product pairs, exhibited dramatic increases in maximal rates, along with decreases in substrate half-saturation values and cooperativity. The carbon kinetic isotope effect, 13C versus 12C at the carbonyl group of carbamoyl phosphate, for the Asp-236-->Ala enzyme decreased toward unity as [Asp] increased, as observed for the wild-type enzyme. Both the kinetic isotope effects and EIEK results indicate that the Asp-236-->Ala enzyme operates by the same ordered kinetic mechanism as the wild-type enzyme. Although activation effects by ATP and N-phosphonacetyl-L-aspartate are lost, inhibition by CTP was apparent in equilibrium exchanges. Simulation of the EIEK data indicated that the best fit to the observed changes in saturation curves was obtained by preferentially increasing the rate of the T-->R transition, kappa T-->R, thereby destabilizing the T-state and increasing the equilbrium constant for the T<-->R transition. A multistep model for Asp bindng to aspartate transcarbamoylase is proposed, in which Asp induces the initial conformational changes that in turn trigger the T-->R transition, followed by stepwise filling of the remaining active sites.

Alanine↗
PubMed1995

Kinetic isotope effects on substrate association: reactions of phosphoenolpyruvate with phosphoenolpyruvate carboxylase and pyruvate kinase.

Kinetic isotope effects on association have been measured using the remote label methodology developed by O'Leary and Marlier (1979). The isotope effect on V/KA for the first substrate in an obligatorily ordered mechanism is an isotope effect on its second-order rate constant for association with the enzyme. With phosphoenolpyruvate carboxylase the 18(V/KPEP) when the bridging O is labeled decreases from 1.0056 +/- 0.0007 to 0.9943 +/- 0.0002 as the concentration of bicarbonate, the second substrate, increases from 2 to 200 mM. With pyruvate kinase the 18(V/KPEP) decreases from 1.0024 +/- 0.0014 to 0.9928 +/- 0.0027 as the concentration of ADP increases from 1.5 to 30 mM. These inverse kinetic isotope effects are best understood as arising from an isotope effect on the rate constant for forming the Michaelis complex of enzyme and substrate. The inverse value suggests that the bridging oxygen is in a vibrationally stiffer environment in the transition state for the association reaction.

Animals↗
PubMed1992

A kinetic investigation of phosphoenolpyruvate carboxylase from Zea mays.

The reaction catalyzed by phosphoenolpyruvate carboxylase from Zea mays has been studied kinetically. Results of initial velocity patterns and inhibition studies indicate that phosphoenolpyruvate carboxylase has a random sequential mechanism in which there is a high level of synergism in the binding of substrates. The preferred order of addition of reactants is Mg2+, phosphoenolpyruvate, and bicarbonate. The binding of Mg2+ is at equilibrium. Values for the various kinetic parameters are KiMg = 2.3 +/- 0.4 mM, KPEP = 3.6 +/- 0.6 mM, KiPEP = 0.2 +/- 0.07 mM, and Kbicarbonate = 0.18 +/- 0.04 mM. In addition, double inhibition experiments have been performed to examine the nature of the active site interactions with the putative intermediates, carboxy phosphate and the enolate of pyruvate. Highly synergistic inhibition of phosphoenolpyruvate carboxylase was observed in the presence of oxalate and carbamyl phosphate (alpha = 0.0013). However, an antisynergistic relationship exists between oxalate and phosphonoformate (alpha = 2.75).

Bicarbonates↗
PubMed1992

Hydrolysis of phosphoenolpyruvate catalyzed by phosphoenolpyruvate carboxylase from Zea mays.

In addition to the normal carboxylation reaction, phosphoenolpyruvate carboxylase from Zea mays catalyzes a HCO3(-)-dependent hydrolysis of phosphoenolpyruvate to pyruvate and Pi. Two independent methods were used to establish this reaction. First, the formation of pyruvate was coupled to lactate dehydrogenase in assay solutions containing high concentrations of L-glutamate and aspartate aminotransferase. Under these conditions, oxalacetic acid produced in the carboxylation reaction was efficiently transaminated, and decarboxylation to form spurious pyruvate was negligible. Second, sequential reduction of oxalacetate and pyruvate was achieved by initially running the reaction in the presence of malate dehydrogenase with NADH in excess over phosphoenolpyruvate. After the reaction was complete, lactate dehydrogenase was added, thus giving a measure of pyruvate concentration. At pH 8.0 in the presence of Mg2+, the rate of phosphoenolpyruvate hydrolysis was 3-7% of the total reaction rate. The hydrolysis reaction catalyzed by phosphoenolpyruvate carboxylase was strongly metal dependent, with rates decreasing in the order Ni2+ greater than Co2+ greater than Mn2+ greater than Mg2+ greater than Ca2+. These results suggest that the active site metal ion binds to the enolate oxygen, thus stabilizing the proposed enolate intermediate. The more stable the enolate, the less reactive it is toward carboxylation and the greater the opportunity for hydrolysis.

Bicarbonates↗
PubMed1992

Mechanistic studies of phosphoenolpyruvate carboxylase from Zea mays with (Z)- and (E)-3-fluorophosphoenolpyruvate as substrates.

The catalytic mechanism of phosphoenolpyruvate (PEP) carboxylase from Zea mays has been studied using (Z)- and (E)-3-fluorophosphoenolpyruvate (F-PEP) as substrates. Both (Z)- and (E)-F-PEP partition between carboxylation to produce 3-fluorooxalacetate and hydrolysis to produce 3-fluoropyruvate. Carboxylation accounts for 3% of the reaction observed with (Z)-F-PEP, resulting in the formation of (R)-3-fluorooxalacetate, and for 86% of the reaction of (E)-F-PEP forming (S)-3-fluorooxalacetate. Carboxylation of F-PEP occurs on the 2-re face, which corresponds to the 2-si face of PEP. The partitioning of F-PEP between carboxylation and hydrolysis is insensitive to pH but varies with metal ion. Use of 18O-labeled bicarbonate produces phosphate that is multiply labeled with 18O; in addition, 18O is also incorporated into residual (Z)- and (E)-F-PEP. The 13(V/K) isotope effect on the carboxylation of F-PEP catalyzed by PEP carboxylase at pH 8.0, 25 degrees C, is 1.049 +/- 0.003 for (Z)-F-PEP and 1.009 +/- 0.006 for (E)-F-PEP. These results are consistent with a mechanism in which carboxylation of PEP occurs via attack of the enolate of pyruvate on CO2 rather than carboxy phosphate. In this mechanism phosphorylation of bicarbonate to give carboxy phosphate and decarboxylation of the latter are reversible steps. An irreversible step, however, precedes partitioning between carboxylation to give oxalacetate and release of CO2, which results in hydrolysis of PEP.

Bicarbonates↗
PubMed1992

Mechanistic studies of phosphoenolpyruvate carboxylase from Zea mays utilizing formate as an alternate substrate for bicarbonate.

Formate is an alternate substrate for bicarbonate in the reaction with PEP catalyzed by phosphoenolpyruvate carboxylase from Zea mays, producing formyl phosphate and pyruvate. The Km for formate is 25 +/- 2 mM, and the maximum velocity is 1% of that for bicarbonate at pH 8.0. Use of [18O]formate produces inorganic phosphate containing 1 equiv of 18O, but no label is incorporated into residual phosphoenolpyruvate. PEP carboxylase catalyzes the hydrolysis of phosphoglycolate or L-phospholactate 2000 times more slowly and D-phospholactate 4000 times more slowly than the reaction between bicarbonate and PEP.

Bicarbonates↗
PubMed1992

13C isotope effects as a probe of the kinetic mechanism and allosteric properties of Escherichia coli aspartate transcarbamylase.

13C kinetic isotope effects have been measured in carbamyl phosphate for the reaction catalyzed by aspartate transcarbamylase. For the holoenzyme, the value was 1.0217 at zero aspartate, but unity at infinite aspartate, with 4.8 mM aspartate eliminating half of the isotope effect. This pattern proves an ordered kinetic mechanism, with carbamyl phosphate adding before aspartate. The same parameters were observed in the presence of ATP or CTP, showing that there is only one form of active enzyme present, regardless of the presence or absence of allosteric modifiers. These data support the Monod model of allosteric behavior in which the equilibrium between fully active and inactive enzyme is perturbed by selective binding interactions of substrates and modifiers, and there are no enzyme forms having partial activity. Isolated catalytic subunits of the enzyme showed similar 13C isotope effects (1.0240 at zero aspartate, 1.0039 at infinite aspartate, 3.8 mM aspartate causing half of the change from one value to the other), but the finite isotope effect at infinite aspartate shows that the kinetic mechanism is now partly random. With the very slow and poorly bound aspartate analog cysteine sulfinate, the 13C isotope effects were 1.039 for both holoenzyme and catalytic subunits and were not decreased significantly by high levels of cysteine sulfinate. The value of 1.039 is probably close to the intrinsic isotope effect on the chemical reaction, while the kinetic mechanism with this substrate is now fully random because the chemistry is so much slower than release of either reactant from the enzyme.

Adenosine Triphosphate↗
PubMed1992

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

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

Aspartate Carbamoyltransferase↗
PubMed1992

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)

Alanine↗
PubMed1992

The contribution of threonine 55 to catalysis in aspartate transcarbamoylase.

Heavy-atom isotope effects and steady-state kinetic parameters were measured for the catalytic trimer of an active site mutant of aspartate transcarbamoylase, T55A, to assess the role of Thr 55 in catalysis. The binding of carbamoyl phosphate to the T55A mutant was decreased by 2 orders of magnitude relative to the wild-type enzyme whereas the affinities for aspartate and succinate were not markedly altered. This indicates that Thr 55 plays a significant role in the binding of CbmP. If, as had been suggested previously, Thr 55 assists in the polarization of the carbonyl group of CbmP, the carbon isotope effect for the T55A mutant should increase relative to that observed for the wild-type enzyme. However, the opposite is seen, indicating that Thr 55 is not involved in stabilizing the oxyanion in the transition state. Quantitative analysis of a series of 13C and 15N isotope effects suggested that the rate-determining step in the reaction catalyzed by T55A trimer may be a conformational change in the protein subsequent to formation of the Michaelis complex. Thus, Thr 55 may facilitate a conformational change in the enzyme that is a prerequisite for catalysis. An altered active site environment in the binary and Michaelis complexes with T55A trimer is reflected in the pH profiles for log V, log (V/K)asp, and pK(i) succinate, show a displacement in the pK values of ionizing residues involved in aspartate binding and catalysis relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartate Carbamoyltransferase↗
PubMed1992

13C isotope effect studies of Escherichia coli aspartate transcarbamylase in the presence of the bisubstrate analog N-(phosphonoacetyl)-L-aspartate.

13C isotope effects have been measured for the aspartate transcarbamylase holoenzyme (ATCase) and catalytic subunit catalyzed reactions in the presence of the bisubstrate analog N-(phosphonoacetyl)-L-aspartate (PALA). For holoenzyme-catalyzed reactions in the physiological direction with very low levels of L-aspartate as substrate, or with L-cysteine sulfinate as substrate, or in the reverse direction with carbamyl-L-aspartate and phosphate as substrates, the isotope effect data show a slight dependence on PALA concentration. Under these conditions, PALA first stimulates the rate and then inhibits it at higher concentrations. The observed isotope effect at maximum stimulation by PALA is slightly smaller than in the absence of the analog, but as the PALA concentration is increased to reduce the rate to its original value, the observed isotope effect also increases and approaches the value of the isotope effect determined in the absence of PALA. These data suggest that the kinetic properties of the active enzyme are affected by the number of active sites occupied by PALA, indicating communication between subunits, and a mathematical model is proposed which explains our experimental observations. In contrast to these results with the holoenzyme, isotope effects measured for the reaction catalyzed by the isolated catalytic subunits are not altered in the presence of PALA. Taken together, these data are consistent with the two-state model for the homotropic regulation of ATCase.

Allosteric Regulation↗
PubMed1992

Binding of carbon dioxide to phosphoenolpyruvate carboxykinase deduced from carbon kinetic isotope effects.

Phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] from Chloris gayana Kunth has been purified by a combination of ammonium sulfate fractionation, ion exchange, gel filtration, and affinity chromatography on agarose-hexane-ATP. In the direction of OAA formation, the specific activity of the enzyme was 33 mumol/(min.mg of protein). The carbon isotope effect on carboxylation was measured by successive analysis of remaining CO2 over the course of the reaction. At 22 mM PEP and 1.3 mM MgADP, pH 7.5, the isotope effect is 1.024 +/- 0.001. When the concentration of PEP was reduced to 1 mM, the isotope effect rose to 1.034 +/- 0.004; when the concentration of MgADP was reduced to 60 microM, the value rose to 1.040 +/- 0.006. The variation of the carbon isotope effect on carboxylation with both substrate concentrations indicates that the enzyme operates by a random kinetic mechanism. This in turn requires that the enzyme have a binding site for substrate CO2; this is one of the first enzymes for which such a site has been demonstrated.

Adenosine Diphosphate↗
PubMed1991

Investigation of the enzymatic mechanism of yeast orotidine-5'-monophosphate decarboxylase using 13C kinetic isotope effects.

Orotidine-5'-monophosphate decarboxylase (ODCase) from Saccharomyces cerevisiae displays an observed 13C kinetic isotope effect of 1.0247 +/- 0.0008 at 25 degrees C, pH 6.8. The observed isotope effect is sensitive to changes in the reaction medium, such as pH, temperature, or glycerol content. The value of 1.0494 +/- 0.0006 measured at pH 4.0, 25 degrees C, is not altered significantly by temperature or glycerol, and thus the intrinsic isotope effect for the reaction is apparently being observed under these conditions and decarboxylation is almost entirely rate-determining. These data require a catalytic mechanism with freely reversible binding and one in which a very limited contribution to the overall rate is made by chemical steps preceding decarboxylation; the zwitterion mechanism of Beak and Siegel [Beak, P. & Siegel, B. (1976) J. Am. Chem. Soc. 98, 3601-3606], which involves only protonation of the pyrimidine ring, is such a mechanism. With use of an intrinsic isotope effect of 1.05, a partitioning factor of less than unity is calculated for ODCase at pH 6.0, 25 degrees C. A quantitative kinetic analysis using this result excludes the possibility of an enzymatic mechanism involving covalent attachment of an enzyme nucleophile to C-5 of the pyrimidine ring. The observed isotope effect does not rise to the intrinsic value above pH 8.5; instead, the observed isotope effects at 25 degrees C plotted against pH yield an asymmetric curve that at high pH plateaus at about 1.035. These data, in conjunction with the pH profile of Vmax/km, fit a kinetic model in which an enzyme proton necessary for catalysis is titrated at high pH, thus providing evidence for the catalytic mechanism of Beak and Siegel (1976).

Carbon Isotopes↗
PubMed1991

Sulfuryl transfer catalyzed by phosphokinases.

Adenosine 5'-sulfatopyrophosphate is a substrate for nucleoside diphosphate kinase. The reaction appears to proceed through a ping-pong mechanism analogous to the physiological reaction involving ATP, presumably by way of a sulfohistidine intermediate. Unlike the phosphoryl transfer reactions, the corresponding sulfuryl transfers catalyzed by nucleoside diphosphate kinase do not have a strict divalent metal requirement. The estimated rate constants for the metal- and nonmetal-catalyzed sulfuryl transfers differ by less than an order of magnitude and are approximately 1000-fold slower than the corresponding phosphate transfers. These results suggest that the role of the metal ion in nucleoside diphosphate kinase is to coordinate the alpha, beta-phosphates of the substrate. Sulfuryl and phosphoryl transfer probably occur through dissociative transition states.

Adenosine Phosphosulfate↗