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H F Fisher

Publications and source records attributed to H F Fisher.

At least 37 records · Page 2Linked to original sources
PubMed1988

Proton/product time course ratios: a new approach to transient-state kinetic analysis.

The usefulness of the indicator dye method for the detection of transient enzyme-product intermediates has been very limited due to the near impossibility of resolving the apparent single exponential time courses resulting from sequences of steps linked by rather similar rate constants. We propose here a novel approach, the proton-product time course method, a procedure which can extract a great deal of the mechanistic information which remains buried in conventional proton release-time course measurements. The method involves nothing more than measuring the ratio, r, of the moles of H+ released to the moles of product formed as a function of time. We derive the theory relating this r function to mechanisms of varying complexity, explore the theoretical behavior of the function in various possible mechanistic situations, and employ the new approach in an experimental system. We demonstrate the fact that the proton/product time course ratio method can provide evidence of the existence of hidden steps in transient state kinetic studies, that it can determine accurate thermodynamic pK values of the intermediate complexes involved in those steps, and that it can produce time courses of individual intermediates which are obscure to conventional kinetic methods.

Coloring Agents↗
PubMed1988

Mechanism of formation of bound alpha-iminoglutarate from alpha-ketoglutarate in the glutamate dehydrogenase reaction. A chemical basis for ammonia recognition.

Two mechanisms have been postulated for the formation of bound alpha-iminoglutarate intermediate during the glutamate dehydrogenase-catalyzed reductive amination of alpha-ketoglutarate; one involves the nucleophilic attack of ammonia on a covalently bound Schiff base in the enzyme-NADPH-alpha-ketoglutarate complex, and the other involves the reaction of ammonia with the carbonyl group of alpha-ketoglutarate in the ternary complex. We have measured the rates of carbonyl oxygen exchange in the complex to unambiguously distinguish between these two mechanisms. We find that the loss of label in the carbonyl oxygen-labeled ternary complex is at least 10(5) times slower than the rate of the reductive amination reaction. Therefore, the former mechanism cannot be operative. We also find that (i) the carbonyl oxygen exchange in free alpha-ketoglutarate proceeds without any significant catalysis by its gamma-carboxylate group; (ii) this exchange reaction has energy parameters which are comparable to those observed for the hydration of simple aliphatic ketones; and (iii) the carbonyl oxygen exchange in bound alpha-ketoglutarate is slower than that in the free keto acid over a wide pH range. We conclude that the oxygen exchange in the free and bound alpha-ketoglutarate must occur via a gem-diol intermediate. The observation that the enzyme inhibits the reaction of water with alpha-ketoglutarate while it catalyzes the reaction of ammonia with the same keto acid points to an extraordinary recognition of ammonia by the enzyme. We interpret this observation by assuming that the enzyme-NADPH-alpha-ketoglutarate complex exists in two forms, a predominant form which is produced rapidly upon mixing the components together and an unstable form which is produced in trace amounts from the predominant form via a gem-diol intermediate. These two forms are presumed to differ in the spatial relationship of the carbonyl group to the enzyme functional groups. The carbonyl group in the unstable form is assumed to be surrounded by the same enzyme groups as the iminium ion is in the bound iminoglutarate complex. We ascribe the remarkable catalysis of the ammonia reaction and the inhibition of the water reaction by the enzyme to the opposing interactions of the iminium and carbonyl groups with these surrounding enzyme groups.

Algorithms↗
PubMed1987

The anomalous properties of the glutamate dehydrogenase-NADPH-alpha-ketoglutarate complex are not ascribable to a carbonyl addition reaction.

The glutamate dehydrogenase-NADPH-alpha-ketoglutarate complex, an active intermediate on the reaction pathway has a number of unusual properties: 1) it is the only blue-shifted natural complex of this enzyme; 2) it has an anomalously slow rate of dissociation; 3) its off-rate shows a substantial pH-independent D2O solvent isotope effect not exhibited by any other ternary complex of this enzyme; and 4) it has an unusually large enthalpy of interaction parameter. These properties must be ascribable to at least one of the two possibilities conferred on the complex by the presence of the alpha-carbonyl group of alpha-ketoglutarate; the ability to engage in carbonyl addition reactions; and/or the ability to form a specific hydrogen bond. Oxalylglycine, a competitive inhibitor of alpha-ketoglutarate in this enzyme-catalyzed reaction, provides a means of discriminating between these two modes of action. The structure of oxalylglycine provides a dicarboxylic compound which has the same intercarboxylate proton distance and has a carbonyl group in a position spatially analogous to that of alpha-ketoglutarate. Its carbonyl group, however, is that of an amide group and cannot, therefore, engage in carbonyl addition reactions, but can hydrogen bond. Therefore, any effects observed with both oxalylglycine and alpha-ketoglutarate must be ascribed to formation of specific alpha-carbonyl hydrogen bonding, whereas any effects observed with alpha-ketoglutarate alone must be due to an alpha-carbonyl addition reaction. We have used this logic to test the source of the four phenomena listed above. In each case, oxalylglycine and alpha-ketoglutarate showed the same effect. Therefore, we conclude that all four phenomena are in fact due to the formation of a specific alpha-carbonyl hydrogen bond and that the specific carbonyl addition reaction between alpha-ketoglutarate and an enzyme lysine group, postulated in one proposed catalytic mechanism, does not occur.

Animals↗
PubMed1987

The effects of an acetate-sensitive anion binding site on NADPH binding in glutamate dehydrogenase.

The nature of a general anion binding site that regulates NADPH binding to L-glutamate dehydrogenase has been explored. Dissociation constants for the enzyme-NADPH complex were measured by difference spectroscopy in the presence of phosphate, pyrophosphate, ADP and acetate ions. Whereas two molecules of phosphate, binding in a cooperative fashion, raise the Kd of the enzyme-NADPH complex 50-fold from 2.3 microM, a single pyrophosphate raises the Kd only 23-fold, disproving the notion that the anion binding site is simply the pyrophosphate binding site of NADPH. ADP raises the Kd of the enzyme-NADPH complex 2-fold for a given phosphate concentration, and formation of the enzyme-ADP complex is itself interfered with by phosphate and pyrophosphate, indicating that these anions interact with the same anion binding site. Acetate ion acts in a manner opposite to that of phosphate, pyrophosphate and ADP and reverses the weakening effect that these ions exert on NADPH binding, returning the Kd of the enzyme-NADPH complex to 2.3 microM. In the absence of these anions, however, acetate exerts no measurable effect on the Kd, suggesting an allosteric mechanism.

Acetates↗
PubMed1986

NADPH binding induced proton ionization as a cause of nonlinear heat capacity changes in glutamate dehydrogenase.

Functional group interactions involved in the formation of the glutamate dehydrogenase-NADPH binary complex have been studied by three independent but complementary approaches: the pH dependence of the overall dissociation constant measured by an improved differential spectroscopic technique; the pH dependence of the enthalpy of complex formation measured by flow calorimetry; and the pH dependence of the number of protons released to, or taken up from, the solvent in the complex formation reaction, measured by titration. We conclude that the coenzyme binds to the enzyme through three distinguishable interactions: a pH-independent process involving the binding of the reduced nicotinamide ring; a relatively weak "proton-stabilizing" process, occurring at low pH involving the shift at a pK of 6.3 in the free enzyme to 7.0 in the enzyme-NADPH complex; and a stronger "proton-destabilizing" process, occurring at a higher pH involving a shift of a pK of 8.5 in the enzyme down to 6.9 in the enzyme-NADPH complex. The proton ionization of the free enzyme involved in this third interaction exhibits some unusual thermodynamic parameters, having delta Go = +11.5 +/- 0.1 kcal mol-1, delta Ho = +19 +/- 1 kcal mol-1, and delta So = +23 eu. We show here that this proton ionization step is directly related to and indeed constitutes the "implicit" shift in enzyme macrostates which we have shown to be responsible for the existence of large highly nonlinear delta Cpo effects in the formation of this complex [Fisher, H. F., Colen, A. H., & Medary, R. T. (1981) Nature (London) 292, 271-272].

Animals↗
PubMed1986

Structural features facilitating the glutamate dehydrogenase catalyzed alpha-imino acid-alpha-amino acid interconversion.

Glutamate dehydrogenase catalyzes the reversible oxidation of L-proline and L-pipecolic acid to the corresponding cyclic alpha-imino acids. The active substrates are the amino acid anion in one direction and the iminium ion in the other. The oxidation of the ester, amide, and N-methyl derivatives of L-proline by enzyme-NADP+ and the reduction of N-methyl-delta 1-tetrahydropyridinium ion by enzyme-NADPH do not proceed to a detectable extent under the experimental conditions. The methyliminium ion, however, undergoes facile nonenzymatic reduction by NADPH. If it is assumed that the nonenzymatic reaction reflects the structural requirements of the redox step of the enzymatic reaction, then the lack of reactivity of the tetrahydropyridinium ion toward enzyme-NADPH must be due to the instability of the central complex. It appears that the alpha-carboxylate and NH groups in the amino acid anion and in the alpha-imino acid are involved in binding the substrates to the enzyme-coenzyme complexes. We conclude that each of these active substrates binds to its appropriate enzyme-coenzyme complex through a hydrogen bond between its NH group and a basic enzyme group; there is also an ionic bond in the central complex between the alpha-carboxylate group of the active substrate and a positively charged enzyme group. The five-membered amino acid anions are more reactive toward enzyme-NADP+ than the six-membered ones. The same reactivity order is seen for the reduction of imino acids by enzyme-NADPH. Since these effects are also present in the nonenzymatic reduction by NADPH we ascribe the ring size effects on V/Ksubstrate primarily to those on the hydride-transfer step.

Amino Acids↗
PubMed1985

Comparison of the energetics of the uncatalyzed and glutamate dehydrogenase catalyzed alpha-imino acid-alpha-amino acid interconversion.

The thermodynamic and activation parameters for the reduction of delta 1-pyrroline-2-carboxylic acid (an alpha-imino acid) by reduced nicotinamide adenine dinucleotide phosphate (NADPH) are compared with those for the reduction of the same imino acid by the glutamate dehydrogenase-NADPH complex. The enthalpies of activation and standard free energy changes for these two reactions are found to be virtually the same. The catalysis by the enzyme, expressed as the ratio of the reactivity of the enzyme--NADPH complex to that of NADPH itself in reducing the iminium ion, is entirely accounted for by a more favorable entropy of activation with enzyme--NADPH as the reductant. This entropic driving force is large enough to overcome the exergonic formation of the binary complex and still lead to considerable catalysis by glutamate dehydrogenase. Comparison of delta S not equal to and delta So values for the reduction of the iminium ion by NADPH suggests that the solvation of the transition state resembles that of the reactants, even though the substituent effects on rate have shown that the hydride transfer from the reduced coenzyme is complete at the transition state [Srinivasan, R., Medary, R. T., Fisher, H. F., Norris, D. J., & Stewart, R. (1982) J. Am. Chem. Soc. 104, 807]. The delta Go and delta S not equal to/delta So values for the reduction by the enzyme--NADPH complex indicate that this reaction has a fairly symmetric transition state, the solvation properties of which are intermediate between those of the reactants and those of the products.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Reversible reduction of an alpha-imino acid to an alpha-amino acid catalyzed by glutamate dehydrogenase: effect of ionizable functional groups.

The glutamate dehydrogenase catalyzed reduction of delta 1-pyrroline-2-carboxylic acid (PCA; an alpha-imino acid) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) to give L-proline and NADP+ is employed as a model for the redox step of the corresponding enzyme-catalyzed reductive amination of alpha-ketoglutarate. We demonstrate the reversibility of the model reaction and measure its equilibrium constant. The pH profiles for the model reactions show that the active substrates are the N-protonated imino acid in one direction and the proline anion with a neutral amino group in the other. The V/K value for the imino acid reduction is enhanced by a group Z of pK = 8.6 in the enzyme-NADPH complex, while that for the proline reaction is unaffected by any such group in the enzyme-NADP+ complex. The following conclusions emerge from a comparison of the pH dependence of the rates for the model reactions with that for the oxidative deamination of L-glutamate [Rife, J. E., & Cleland, W. W. (1980) Biochemistry 19, 2328]. The N-protonated form of alpha-iminoglutarate and the conjugate base of glutamate are the active substrates. The redox step is not sensitive to the protonation state of the groups that catalyze the hydrolysis of bound alpha-iminoglutarate. The group Z, which facilitates the PCA reaction, plays no role in the binding of alpha-ketoglutarate. We propose a chemical mechanism for the glutamate reaction where an unprotonated enzyme group of pK = 5.2 in enzyme-NADPH catalyzes the conversion of the alpha-iminoglutarate to the carbinolamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Thermodynamic interactions in the glutamate dehydrogenase-NADPH-oxalylglycine complex.

The enzyme-reduced coenzyme-alpha-ketoglutarate ternary complex is a critical intermediate in the glutamate dehydrogenase-catalyzed reaction. Oxalylglycine, a structural analog of alpha-ketoglutarate which contains an amide carbonyl group in place of a reducible ketone group, is one of the few compounds known to complete with alpha-ketoglutarate itself. In order to examine the role of the ketone group of alpha-ketoglutarate in the ternary complex, we have carried out a calorimetric study of the corresponding oxalylglycine ternary complex, determining the complete delta H, delta G, delta S, and delta Cp profiles and the corresponding interaction parameters for that complex and have compared the various parameters with the corresponding ones previously reported for the alpha-ketoglutarate ternary complex. While the overall delta G values of the two ternary complexes differ only slightly, the enzyme-NADPH-oxalylglycine ternary complex appears to achieve much of its stability from a very tight enzyme-oxalylglycine binary complex with little or no contribution from favorable interactions in the ternary complex, while the alpha-ketoglutarate ternary complex appears to achieve the same stability by a large interaction starting from a very weak enzyme-alpha-ketoglutarate binary complex. Consideration of the enthalpic profiles, however, show that this delta G-derived picture is deceptive. The excess binding energy which stabilizes the oxalylglycine binary is in fact due to hydrogen bonding of the amide group of oxalylglycine to the enzyme; in forming the ternary complex, this hydrogen bonding is lost in favor of forming an oxalylglycine-NADPH interaction, which is very similar to the alpha-ketoglutarate-NADPH interaction which stabilizes the alpha-ketoglutarate ternary complex. We conclude that the alpha-ketoglutarate-NADPH interaction must depend on either hydrogen bonding to or steric hindrance by the ketone group and that the existence of this energetically large interaction cannot be ascribed to imine formation between the keto group and enzyme. These findings also indicate the locus on the reaction coordinate where the reduced coenzyme plays a critical role, a role other than its obvious function as a hydride donor.

Amino Acids, Dicarboxylic↗
PubMed1984

Carbonyl oxygen exchange evidence of imine formation in the glutamate dehydrogenase reaction and identification of the "occult role" of NADPH.

Although an imine intermediate has long been postulated as participating in the reaction catalyzed by glutamate dehydrogenase (EC 1.4.1.4), direct evidence for a kinetically competent intermediate of this kind has not heretofore been found. We have sought such evidence by studying the exchange of the carbonyl oxygen atom of alpha-ketoglutarate in a variety of binary, ternary, and quaternary enzyme complexes. We have found that the time course of this exchange is biphasic when the enzyme, alpha-ketoglutarate, NADPH, and ammonia are all present initially and that the rapid initial phase ends when ammonia is depleted. We present evidence that this rapid exchange is due to an imine form of the enzyme-reduced-coenzyme-substrate-ammonia complex. Formed very rapidly but in very small amounts, this imine can undergo one of two competing fates: (i) hydrolytic reversal to form carbonyl-exchanged alpha-ketoglutarate with regeneration of ammonia, and (ii) an internal hydride transfer converting the iminoglutarate to glutamate, whereby ammonia is consumed. The agreement of the amplitudes of rapid 18O exchange with predictions based on direct transient-state spectroscopic kinetic studies supports the identity of an enzyme-NADPH-alpha-iminoglutarate complex as an obligatory intermediate on the enzyme-catalyzed reaction path. The corresponding enzyme-alpha-iminoglutarate binary complex (previously suggested as an intermediate) is formed at a rate that is less than 1/1000th of that of the NADPH-containing complex shown here, and it therefore lacks kinetic competence. This finding points up an important catalytic role for NADPH that does not involve its obvious function as a hydride donor and is distinctly separate from this role. In the case of the glutamate dehydrogenase-catalyzed reaction, this "occult role" clearly involves the induction of ketimine formation on the enzyme surface.

Ammonia↗
PubMed1983

Configurational, conformational, and solvent effects on the reduction of a Schiff base by reduced pyridine nucleotide analogs.

The rate of reduction of the Schiff base, delta 1-pyrroline-2-carboxylic acid, by beta-NADH has previously been shown to be 6.8 times smaller than that calculated from the substituent effects in 1-substituted 1,4-dihydronicotinamides. The factors governing this anomalous rate have been studied by measuring the reduction rate with a number of reduced pyridine nucleotides in water and water-methanol mixtures. The beta-configuration for the nicotinamide-ribosidic linkage was found to be responsible for 75% of the anomaly; the same reduced coenzyme with alpha-linkage, however, behaves normally. It was also shown that the low reactivities of the reduced coenzymes with beta-configuration are entirely the result of their abnormal thermodynamic stabilities. Conformational effects (the folding of beta-NADH) account for only 20% of the reduction rate anomaly. Addition of organic solvents causes only a modest decrease in the overall rate. These solvent effects are interpreted in terms of the opposing effects of solvents on the basicity of the Schiff base and on the reduction step itself. Specific solvation effects appear important in controlling the reduction rates.

Kinetics↗
PubMed1982

Effect of cryosolvent on transient kinetics of the glutamate dehydrogenase reaction.

The transient and steady-state kinetics of the oxidative deamination of L-glutamate by glutamate dehydrogenase and NADP in both aqueous solution and 30% methanol are compared. Methanol causes an approximately 5-fold tightening of the enzyme--L-glutamate binary complex and an approximately 2-fold reduction of the interaction parameter for the ternary enzyme--NADP--L-glutamate complex. The most dramatic effect of methanol on the time course of the reaction is what appears to be a conversion of the enzyme at substoichiometric initial levels of reactant NADP to a form from which product alpha-ketoglutarate does not readily dissociate. This conversion appears only at NADP concentrations over one-third of the enzyme active site concentration.

Animals↗
PubMed1982

Glutamate dehydrogenase catalyzes the reduction of a Schiff base (delta 1-pyrroline-2-carboxylic acid) by NADPH.

alpha-Iminoglutarate has long been postulated as an obligatory intermediate in the glutamate dehydrogenase catalyzed reaction, but direct proof of its participation is lacking. We report here the glutamate dehydrogenase catalyzed reduction of delta 1-pyrroline-2-carboxylic acid (a cyclic-alpha-imino acid) to proline (an alpha-amino acid). The catalysis occurs at the normal catalytic site of the enzyme. The imine and the enzyme-NADPH complex are the active oxidant and reductant, respectively. The latter is about 500 times more reactive than NADPH itself. These findings provide direct evidence that the glutamate dehydrogenase catalyzed reaction does indeed proceed by way of an enzyme-bound form of an alpha-iminocarboxylic acid.

Animals↗
PubMed1982

alpha-Ketoglutaric acid: solution structure and the active form for reductive amination by bovine liver glutamate dehydrogenase.

A study of the various solution forms of alpha-ketoglutaric acid using UV absorption spectrophotometry and 13C NMR spectroscopy shows that at neutral pH alpha-ketoglutarate exists predominantly as the keto form with about 7% hydrated form (gem-diol) and a small amount of cyclic form. Protonation of the gamma-carboxylate group increases the amount of cyclic form to 20% with very little increase in the amount of gem-diol. Protonation of the alpha-carboxylate increases the amount of cyclic form to 30% and the amount of gem-diol to 35%. The pH and temperature dependence of the 13C NMR line widths indicated that the interconversion of keto and cyclic forms is extremely rapid. The rate of interconversion of keto and gem-diol forms was studied spectrophotometrically at various temperatures, buffers, and pH values and by varying the total concentration of alpha-ketoglutarate. The hydration reaction of alpha-ketoglutarate is not catalyzed by bovine liver glutamate dehydrogenase (E) or by the E-NADPH complex. The enzyme uses the keto form of alpha-ketoglutarate as the substrate. The gem-diol form is not itself a substrate but becomes converted to the keto form with a half-life of about 0.3 min at 15 degrees C in 0.1 M potassium phosphate buffer (pH 7.6).

Animals↗
PubMed1981

Temperature-dependent delta C0p generated by a shift in equilibrium between macrostates of an enzyme.

Substantial negative heat capacity changes (delta C0p' s) have frequently been observed to accompany the formation of protein-ligand complexes. Glutamate dehydrogenase and horse liver alcohol dehydrogenase, however, have been reported to form binary complexes with coenzyme with negligible delta H0p' and only small delta C0p' s. Although many intriguing mechanisms have been proposed to account for the observed phenomena, there is little direct experimental evidence available which might provide a basis for evaluating the contributions of delta C0p' s of complex formation from the various mechanistic sources or even for distinguishing between them. However, if, as Eftink and Biltonen have suggested, a shift in equilibrium between macrostates contributes significantly to an observed delta C0p' s for a given reaction, it should be possible to characterize such a system by measuring the temperature dependence of the delta C0p'. Despite this, few studies have determined delta H0' values at more than two temperatures. We have now measured the temperature dependence of the delta H0' (and, thereby, that of the delta C0p') of the formation of an enzyme-reduced coenzyme complex in an attempt to provide such a basis and have found that the entire delta C0p' of complex formation is accounted for by a temperature-induced shift of an equilibrium between the different forms of the free enzyme.

Glutamate Dehydrogenase↗