Abstract
Abstract The activation of NADH- and NADPH-dependent glutamate dehydrogenase-catalyzed reactions appears to be similar for two structurally different compounds, ADP and any of a series of monocarboxylic l-α-amino acids. ADP and l-leucine have identical effects on a large variety of glutamate dehydrogenase-catalyzed reactions. Transient state studies show that l-leucine increases the rate of release of NADPH from an enzyme-NADPH complex in a manner similar to that of ADP. The dissociation constant for the glutamate dehydrogenase-NADPH complex as measured by difference spectroscopy is increased by leucine, indicating that leucine interferes with this mode of NADPH binding. It is shown that the dissociation constant for this leucine complex which interferes with a mode of NADPH binding is equal to the reported dissociation constant for the binary enzyme-leucine complex. The steady state kinetic activation constant which can be obtained by extrapolation to infinitely dilute substrate concentrations is equal to the dissociation constant of the binary enzyme-leucine complex. Since the tight binding of NADPH measured spectroscopically in the 340 nm region appears to be involved in a rate-limiting step, the mechanism of activation of α-keto-glutarate reduction by ADP and monocarboxylic l-α-amino acids may be to weaken this NADPH binding mode. Although kinetic activation data and the binding interactions with NADPH are similar for ADP and l-leucine, each activator enhances the binding of the other and, together, they have a slightly additive activation. Spectroscopic data presented in this paper indicate that the two types of activators have spatially separate binding sites on the enzyme and interact in a positive manner to enhance the binding of each other (i.e. positive cooperativity).
Highlights
The activation of NADH- and NADPH-dependent gluta- mechanism of activation by the monocarboxylic amino acids and mate dehydrogenase-catalyzed reactions appearsto be simi- ADP has been assumed to be the same (2)
Cross and Fisher (4) further increased by leucine, indicating that leucine interferes with characterized the activation of glutamate dehydrogenase reacthis modeof NADPH binding. It is shownthat the dissocia- tions and the interference of NADPH binding by ADP. It was tion constant for this leucine complex which interferes with a shown that ADP reversed GTP inhibition and partially reversed mode of NADPH binding is equalto the reported dissociation L-glutamate inhibition of a-ketoglutarate reduction
The steady activation was seen when 3-acetylpyridine adenine dinucleotide state kinetic activation constant which can be obtained by was employed as the pyridine nucleotide substrate, but ADP did extrapolation to infinitely dilute substrate concentrations is reverse the GTP inhibition of the glutamate dehydrogenaseequal to the dissociationconstant of the binary enzyme-leu- catalyzed reaction of 3-acetylpyridine adenine dinucleotide
Summary
The activation of NADH- and NADPH-dependent gluta- mechanism of activation by the monocarboxylic amino acids and mate dehydrogenase-catalyzed reactions appearsto be simi- ADP has been assumed to be the same (2). Since the tight binding of NADPH measured the oxidation of n-glutamate with a poor coenzyme anaspectroscopicallyin the 340 nm region appearsto be involved logue, NMN+, was inhibited by ADP These data are consistent in a rate-limiting step, the mechanismof activation of ar-keto- with the view that ADP affects the steady state rate of some glutarate reduction by ADP and monocarboxylic L-o-amino glutamate dehydrogenase-catalyzed reactions by decreasing the acids may be to weaken this NADPH binding mode. We have established spectrophotometrically the extions with NADPH are similar for ADP and L-leucine, each istence and specificity of a number of L-glutamate dehydrogenaseactivator enhances the binding of the other and, together, monocarboxylic L-a-amino acid complexes (5, 6) Preliminary they have a slightly additive activation.
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