Harvey F. Fisher

Transient-state intermediates involved in the hydride transfer step of the glutamate dehydrogenase reaction☆

Abstract Stopped-flow spectrophotometric studies of the oxidative deamination of L-glutamate by L-glutamate dehydrogenase and TPN show that the rapid first phase or “burst” in absorbance is due to the formation of a previously unreported complex with a maximum absorbance at 332 mμ. The second slower phase consists of the maintenance of a constant level of this blue-shifted complex accompanied by a slow production of complex with a peak at about 348 mμ. Free TPNH release occurs only later as a third stage. Substitution of deuterium for the α-H atom of L-glutamate produces an isotope effect on the first slope of the reaction, but no effect on the burst height or later phases of the reaction. It is concluded that the burst phase represents the hydride transfer step of the reaction and that the 332 mμ peak is probably the resulting enzyme-TPNH-α-ketoglutarate-NH 4 + complex.

The binding of α-ketoglutarate in a binary complex and in a ternary complex with NADP+ by l-glutamate dehydrogenase

l-Glutamate dehydrogenase (l-glutamate: NAD(P)+ oxidoreductase (deaminating EC 1.4.1.3)), cooperatively binds NADP+ and α-ketoglutarate in a highly stable dead-end complex exhibiting a near ultraviolet difference spectrum characteristic of red shifts of tryptophan and oxidized nicotinamide absorbance. The requirements of an intact amide on the micotinamide moiety and two carboxyl groups on the substrate for a 200-fold heterotropic cooperativity in binding are demonstrated by the use of coenzyme and substrate analogs. n nThe agreement of the binary complex dissociation constants calculated from the concentration dependence of the formation of the abortive complex with those determined directly shows that both coenzyme and α-ketoglutarate are bound at the same site in the binary and ternary complexes. By analogy it is inferred that NADP+ and l-glutamate bind to the enzyme to form the active complex with a cooperativity similar to that demonstrated for the dead-end complex. n nThe ability of the enzyme to form the NADP+ -α-ketoglutarate dead-end complex and other stable complexes is pertinent to the catalytic mechanisms proposed for glutamate dehydrogenase. This ability also provides a mechanism through which both the in vivo direction and rate of catalysis can be selectively and sensitively controlled by the cooperative binding of the reactants and products themselves.

Enzyme-Coenzyme Complexes of Pyridine Nucleotide-Linked Dehydrogenases

Enzyme-reduced coenzyme binary complexes produce previously unreported shifts in the spectrum of the free coenzyme. These shifts give rise to difference spectra which resemble a general environmental change for reduced diphosphopyridine nucleotide (DPNH) in the glutamic dehydrogenase-DPNH complex, and indicate a more specific enzyme-coenzyme interaction for yeast alcohol dehydrogenase-DPNH, isocitrate dehydrogenase-TPNH, and lactic dehydrogenase-DPNH complexes.

Spectrophotometric observation of a glutamate dehydrogenase-l-glutamate complex

Abstract Ultraviolet differential spectroscopic measurements show the existence of a glutamate dehydrogenase ( l -glutamate:NAD(P) + oxidoreductase (deaminating), EC 1.4.1.3)— l -glutamate complex. The spectral features resemble perturbation difference spectra of enzyme aromatic amino acid chromophores and allow the determination of the l -glutamate concentration dependence. The dissociation constant for this enzyme- l -glutamate complex was approximately 48 mM and was independent of enzyme concentration. The lack of interaction between the binding of l -glutamate and the activating monocarboxylic amino acids indicates that they bind at totally separate sites.

Spectrophotometric evidence for a glutamate dehydrogenase-l-leucine complex

Abstract Ultraviolet differential spectroscopic measurements show the existence of a glutamate dehydrogenase- l -leucine complex. Similar complexes can be formed with other amino acids. The dissociation constant for the enzyme- l -leucine complex was 270 μ m and was independent of the state of association of the enzyme. l -Leucine had little effect on the association state of the enzyme. The formation of this enzyme-ligand complex appears to be related to the ability of several amino acids to activate the glutamate dehydrogenase reaction.

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.

The independence of adenosine-5′-diphosphate binding and the state of association of L-glutamate dehydrogenase

Summary The dissociation constant of the adenosine-5′-diphosphate (ADP)-glutamate dehydrogenase complex in the absence of coenzyme was found to be independent of glutamate dehydrogenase concentration and ADP has no appreciable effect on the association state of the enzyme in the absence of coenzyme. These results indicate that ADP does not bind preferentially to any one form of the enzyme and does not by itself affect the reversible, protein concentration-dependent polymerization of the enzyme. The reversal of the effect of dissociative agents such as DPNH and GTP in the presence of DPNH may be explained by their displacement by ADP.

An intermediate in the pH-induced dissociation of glutamate dehydrogenase.

Abstract We have carried out light-scatter and spectrophotometric stopped-flow studies on the acid induced dissociation of bovine liver L-glutamate dehydrogenase. The light-scatter intensity and the absorbance due to chromophore exposure follow the same time dependence, both changing only after a time lag of about 40 msec. The kinetics are consistent with a simple consecutive, two-step, irreversible reaction in which the first step converts β subunits into other forms identical in molecular weight and degree of folding and is followed by a second step in which these altered forms are converted into random coil, fully dissociated peptide chains.

Determination of carbonyl oxygen exchange rates in α-ketoacids by gas chromatography-mass spectrometry

Abstract A method for determining the rate of exchange of the carbonyl oxygen in α-ketoglutarate using gas chromatography-mass spectrometry is described. The method is based on a new procedure for quenching the exchange reaction by rapid oxidative decarboxylation of the α-ketoacid to the next lower homologous carboxylic acid using concentrated hydrogen peroxide. Using this method the rate constant for carbonyl oxygen exchange in α-ketoglutarate in 0.1 m imidazole buffer, pH 7.5, was found to be (2.8 ± 0.2) × 10−3 s−1 at 25°C. Data obtained using this technique suggest that hydration is the mechanism for carbonyl oxygen exchange in α-ketoacids. The method is also applicable to the measurement of oxygen exchange rates of other α-ketoacids free in solution and bound in enzyme complexes.