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H F Fisher

Publications and source records attributed to H F Fisher.

At least 55 records · Page 3Linked to original sources
PubMed1981

Evidence for a change in the structure of glutamate dehydrogenase during its catalytic cycle under cryogenic conditions.

Glutamate dehydrogenase binds alpha-ketoglutarate and NADPH to form a ternary complex whose ultraviolet difference spectrum exhibits a blue-shifted coenzyme absorption band and a distinctive aromatic amino acid perturbation. When ammonia is added to this complex at -42 degrees C in 50% methanol, initiating the enzymatic reaction, these two spectral features disappear at different rates. The kinetic independence of these two features is especially evident in the presence of excess L-glutamate. We propose that, under cryogenic conditions at least, there are two forms of the enzyme. The blue shift of the coenzyme absorption band reflects only the physical presence of an alpha-ketoglutarate molecule at the active site, while the distinctive aromatic amino acid perturbation reflects a change in enzyme structure caused by alpha-ketoglutarate binding which may persist in the absence of any bound alpha-ketoglutarate molecule. Simple red and blue shifts of model tyrosine and tryptophan compounds cannot be used to simulate the observed aromatic amino acid perturbation.

Acetates↗
PubMed1980

pH-dependent thermodynamic parameters of the glutamate dehydrogenase-alpha-ketoglutarate-NADPH complex.

The enthalpy of formation of the reactive bovine glutamate dehydrogenase-alpha-ketoglutarate-NADPH complex has been measured as a function of pH, at two temperatures and in two buffer systems having different enthalpies of ionization. The results demonstrate the existence of an extensive two-way traffic of protons between the buffer and the complex itself. While the pattern in a single buffer is too complex to resolve within the experimental limitations imposed by the system, we develop a theory which shows that the difference in delta H0H+ between buffers of different enthalpies of ionization has a simpler, more easily resolvable form. From such a relationship we show that in the process of binding of a molecule of NADPH to the enzyme-alpha-ketoglutarate complex, a proton has been shifted from the buffer to the complex and that this is due to the shift of the pK of an ionized group on the enzyme to a much higher value when the complex is formed. The delta Cp of formation of the complex is -450 cal . deg.-1 . M-1, and is pH independent. Implications of these results for the interpretation of enthalpies of enzyme-complex formation obtained at a single pH, and at one temperature, with one buffer system are mentioned. The complexity of the pattern of the dependence of delta H0, taken together with the magnitudes of the individual features of these dependencies, suggest that a number of large pK shifts occur when the enzyme-NADPH-alpha-ketoglutarate complex is formed. It is quite probable that these shifts represent a sizeable fraction of the machinery of the catalytic mechanism.

Animals↗
PubMed1980

The thermodynamics of a negatively interacting allosteric effector system. The glutamate dehydrogenase . NADPH . ADP complexes.

The reaction catalyzed by bovine L-glutamate dehydrogenase is subjected to allosteric activation by ADP. We have measured the thermodynamic parameters (delta G0, delta H0, delta S0, and delta Cp) of the formation of the various possible binary and ternary complexes formed between the enzyme, NADPH, and either ADP or its analogs, adenosine, AMP, and ATP. delta H0 and delta Cp have been measured by flow calorimetry; delta G0 values obtained by calorimetry itself, differences spectroscopy, or gel filtration have been selected on the basis of accuracy under the conditions required for the formation of each complex. The data are interpreted in terms of "interaction parameters," the differences between the thermodynamic parameters of the formation of a ternary complex and the sum of those of the two related binary complexes. Both adnosine and ATP appear to loosen the binding of NADPH by simply preventing a subsite interaction of NADPH. AMP appears to have only minor and probably secondary effects. The negative effect of the binding of ADP on that of NADPH, however, involves the formation of a new interaction, which is exothermic, entropy compensated, has a moderately large negative delta Cp, and does not occur in either binary complex.

Adenosine↗
PubMed1979

Effect of ammonia on the glutamate dehydrogenase catalyzed oxidative deamination of L-glutamate. The steady state.

Ammonia is known to inhibit the steady-state rate of oxidation of L-glutamate catalyzed by glutamate dehydrogenase. We reported previously [Brown, A., Colen, A. H., & Fisher, H. F. (1978) Biochemistry 17, 2031] kinetic evidence supporting the formation in the initial rapid phase of a complex which is composed of enzyme, reduced coenzyme, alpha-ketoglutarate, and ammonia. We show here that the effects of ammonia on the steady-state reaction can be correlated with transient-state kinetic effects related to the concentration of that ammonia-containing complex. These results indicate the existence of alternate reaction pathways which become important at high ammonia concentrations. These new pathways provide an additional route for the release of NADPH from the enzyme surface. The expanded mechanism shows that the noncompetitive product inhibition by ammonia can occur without the simultaneous presence of ammonia and L-glutamate on the enzyme. This mechanism also accommodates the observed substrate inhibition by L-glutamate.

Ammonia↗
PubMed1979

The effects of methanol on the glutamate dehydrogenase reaction at 0 degrees C.

The effects of 0-30% methanol (vol/vol) on the Km an Vm values for both the forward and reverse directions of the L-glutamate dehydrogenase reaction were determined at 0 degrees C. The decrease in temperature alone had very little effect on these parameters. However, in the forward reaction, 30% methanol resulted in a 14-fold decrease in the Km value for glutamate, a slight decrease in the Km value for NADP, and a thirty-fold decrease in Vm. Substrate inhibition by glutamate was observed at concentrations greater than 4 mM. In the reverse reaction, 30% methanol caused a decrease in the Km values for alpha-ketoglutarate and ammonia and a 10-fold decrease in Vm. Substrate inhibition by both alpha-ketoglutarate and NADPH was observed at concentrations of either substrate above 0.03 mM. The dependence of Km for glutamate and Vm values for the forward reaction on methanol concentration suggests that they are similarly affected by methanol, in direct contrast to results obtained for NADP. Methanol appeared to cause a general tightening of complexes, which may arise from an effect on the "activities" of species in solution. The use of methanol not only allows for the study of reaction intermediates by slowing the reaction with the cryogenic method, but may also serve as a mechanistic probe by affecting several polarity as well as Km, Vm, and K1 values.

Animals↗
PubMed1978

Effect of ammonia on the glutamate dehydrogenase catalyzed oxidative deamination of L-glutamate: production of an ammonia-containing intermediate in the "burst" phase.

We have studied the effects of ammonium acetate on the transient "burst" phase of the oxidation of L-glutamate by glutamate dehydrogenase. Two measurable changes are observed in the "burst" phase as ammonium acetate concentration is increased: (i) an increase in the apparent first-order rate constant, kapp, and (ii) a decrease in the amplitude of the absorbance change measured at 320 nm. The increase in kapp shows a hyperbolic dependence on ammonium acetate concentration and is independent of glutamate concentration. The results demonstrate the existence of an intermediate immediately following hydrogen transfer. The intermediate contains enzyme, reduced coenzyme, ammonia, and alpha-ketoglutarate moieties and is in equilibrium with the known complex consisting of enzyme, reduced coenzyme, and alpha-ketoglutarate. At high concentrations of ammonium acetate, the equilibrium favors the ammonia containing complex.

Ammonia↗
PubMed1977

The transient-state kinetics of L-glutamate dehydrogenase. pH-dependence of the burst rate parameters.

The pH dependence of the initial transient velocity of NADPH production during the burst phase of the oxidative deamination of L-glutamate by L-glutamate dehydrogenase (L-glutamate : NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) and NADP+ has been measured by stopped-flow spectrophotometry. These studies provide evidence that the entire pH dependence below pH 8.26 arises from reaction steps contributing to V of the burst with an apparent pKa of 8.1 +/- 0.1. The data are consistent with a model in which the formation of the first enzyme-coenzyme-substrate ternary complex on the reaction path equilibrates rapidly and in which the pH-dependent steps are mechanistically close to and may include the catalytic hydrogen transfer itself. At pH 8.87, there is evidence that L-glutamate binds less tightly to the enzyme and to the enzyme-NADP+ complex than at lower pH values.

Animals↗
PubMed1976

Hydrogen exchange at the amide group of reduced pyridine nucleotides and the inhibition of that reaction by dehydrogenases.

Stopped flow ultraviolet spectroscopy has been used to measure the rate of hydrogen exchange with solvent at the amide group of reduced nicotinamide nucleotide coenzymes. Several mechanisms for the exchange reaction are considered in the light of the kinetic data. Complex formation between the coenzyme and any of four dehydrogenases markedly slows the rate of hydrogen exchange. Hydrogen bond formation and/or hydrophobic interactions within these complexes are thought to be the reasons for the decreased rate of exchange.

Alcohol Oxidoreductases↗
PubMed1975

Thermodynamics of complex formation between bovine liver glutamate dehydrogenase and analogs of ADP.

A calorimetric study of the thermodynamic parameters for the binding of adenosine, AMP, ADP, and ATP to L-glutamate dehydrogenase shows that the variation of deltaG0 of binding is quite small and is correlated qualitatively both with the effectiveness of these ribonucleotides as activators of the L-glutamate dehydrogenase reaction and with size (for the first three). Much larger variations are observed for the deltaH0 of binding largely compensated by changes in deltaS0, with a zig-zag dependence on the number of phosphate groups. For comparison, the binding parameters are also obtained for the deoxyribose analogs of these compounds as well as cyclic adenosine 3':5'-monophosphate and purine riboside. Salt concentration and buffer composition were shown to affect mainly the entropy changes for ADP binding; and the deltaCp values for binding of AMP and ADP to the enzyme are quite small. It is suggested that the general area of the enzyme surface which includes the binding sites for ADP and its analogs contains a number of functional groups, each capable of an energetically small interaction with some group on one or more of the ligands, but so located that even the largest ligand cannot interact with all of them simultaneously. Each ligand minimizes the free energy of the system by selecting the best pattern of such individual interactions permitted by its geometry. Such a pattern of microheterogeneity of ligand-protein interactions may be a major source of the known specificity of binding in biological systems.

Adenosine↗
PubMed1975

Location of deuterium oxide solvent isotope effects in the glutamate dehydrogenase reaction.

Stopped flow studies of D2O kinetic solvent isotope effects on the reaction catalyzed by L-glutamate dehydrogenase reveal, in addition to several effects apparently attributable simply to pKa shifts, a 2-fold pH-independent effect on the velocity of the steady state oxidative deamination of L-glutamate by enzyme and NADP. Comparable pH-independent D2O kinetic solvent isotope effects are seen both in a transient phase of the reaction in which alpha-ketoglutarate is displaced by L-glutamate from an enzyme-NADPH-alpha-ketoglutarate (product) complex and in an analogous model reaction in which alpha-ketoglutarate is displaced by D-glutamate. These results suggest that alpha-ketoglutarate dissociation from an enzyme-NADPH-alpha-ketoglutarate complex is rate-limiting in the steady state.

Deuterium↗
PubMed1975

Hydrogen-deuterium exchange in nucleosides and nucleotides. A mechanism for exchange of the exocyclic amino hydrogens of adenosine.

The pH dependence of the apparent first-order rate constant for the exchange of the exocyclic amino hydrogens of adenosine with deuterium from the solvent was measured by stopped-flow ultraviolet spectroscopy. This dependence shows acid catalysis, base catalysis, and spontaneous exchange at neutral pH values. A study of the effect of several buffers on the rates of exchange reveals both general acid and general base catalytic behavior for the exchange process. We propose a general mechanism for the exchange which requires N-1 protonated adenosine as an intermediate for the acid-catalyzed exchange and amidine anion for the base-catalyzed exchange. In both cases the rate-limiting step is the base-catalyzed abstraction of a proton from the exocyclic amino moiety. Evaluation of the rate constants predicts the equilibrium for the exocyclic amino/imino tautomers to be 6.3 times 10(3):1.

Adenosine↗