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.
D000085:Acetates; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D003080:Cold Temperature; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet
D000085:Acetates; D005971:Glutamates; D007656:Ketoglutaric Acids; D000644:Quaternary Ammonium Compounds; D018698:Glutamic Acid; D009249:NADP; D005969:Glutamate Dehydrogenase; C018824:ammonium acetate