Very little discrimination is observed in the binary binding of dicarboxylic acid substrate analogues to glutamate dehydrogenase as monitored by proton nuclear magnetic resonance. Variation in length, charge, bulkiness and conformational rigidity resulted in only a factor of five variation in KD and apparent relaxation time, Tz.Upon titration of the binary enzyme-ligand complex with coenzyme to form the ternary enzyme-ligand-coenzyme complex strong discrimination is observed. Coenzyme binds tightly only when the correct substrate is present, otherwise it binds 10 to 150 times more weakly.Fisher et al. [1,16] proposed that the activity of glutamate dehydrogenase could be controlled by a complex series of ligand isomerizations involving ligand-ligand contacts rather than by ligand-induced protein isomerizations characteristically associated with conformational change models. The ability to discriminate between these two theories depends on direct observation of ligand-induced ligand isomerizations. Direct observation has previously taken the form of monitoring spectrophotometric characteristics of various complexes or changes in these characteristics upon formation of new complexes. In the case of binary complexes of L-glutamate or 2-oxoglutarate with glutamate dehydrogenase, changes were so small that sophisticated spectrophotometric methods were needed [4,7]. 'In 1974 Andree and Zantema [5] demonstrated that binary and ternary complex formation of glutamate dehydrogenase with 2-oxoglutarate and NADPH could be monitored by nuclear magnetic resonance techniques. The parameters readily obtained from such measurements include dissociation constants and the relaxation time, Tz, which is a measure of the rotational correlation time and the intimacy of the interaction between ligand and enzyme.Since positive heterotropic cooperative interactions have been observed during the transition between the binary and ternary complex, it is essential to define the relationship between the bound ligand in Abbreviations. NMR, nuclear magnetic resonance. Enzyme. Glutamate dehydrogenase or L-glutamate: NAD(P)+ oxidoreductase (deaminating) (EC 1.4.1.3 The studies presented in this report focus on the binding characteristics of the substrate binding site of glutamate dehydrogenase in binary complexes with substrate analogues and the relationship between specificity at this stage versus the ternary complex stage. MATERIALS AND METHODS MaterialsBovine liver glutamate dehydrogenase was obtained as a suspension in ammonium sulphate from Boehringer (Mannheim). The enzyme was dialysed at 4 "C, first against buffer containing 0.05 M TrisHCl, 0.1 M NaCl, 0.05 M EDTA at pH 8.0, then two or three times against the same buffer, pH 7.4, containing 0.1 mM EDTA. For NMR measurements two or three dialyses were performed against the same buffer in 'HzO, pH 7.4 (meter reading). Molar enzyme concentrations were expressed in protomers, with a protomer molecular weight of 56000 [lo]. They were calculated from the absorbance at 280 nm, using an absorption...
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