Carbonyl oxygen exchange evidence of imine formation in the glutamate dehydrogenase reaction and identification of the "occult role" of NADPH.
Although an imine intermediate has long been postulated as participating in the reaction catalyzed by glutamate dehydrogenase (EC 1.4.1.4), direct evidence for a kinetically competent intermediate of this kind has not heretofore been found. We have sought such evidence by studying the exchange of the carbonyl oxygen atom of alpha-ketoglutarate in a variety of binary, ternary, and quaternary enzyme complexes. We have found that the time course of this exchange is biphasic when the enzyme, alpha-ketoglutarate, NADPH, and ammonia are all present initially and that the rapid initial phase ends when ammonia is depleted. We present evidence that this rapid exchange is due to an imine form of the enzyme-reduced-coenzyme-substrate-ammonia complex. Formed very rapidly but in very small amounts, this imine can undergo one of two competing fates: (i) hydrolytic reversal to form carbonyl-exchanged alpha-ketoglutarate with regeneration of ammonia, and (ii) an internal hydride transfer converting the iminoglutarate to glutamate, whereby ammonia is consumed. The agreement of the amplitudes of rapid 18O exchange with predictions based on direct transient-state spectroscopic kinetic studies supports the identity of an enzyme-NADPH-alpha-iminoglutarate complex as an obligatory intermediate on the enzyme-catalyzed reaction path. The corresponding enzyme-alpha-iminoglutarate binary complex (previously suggested as an intermediate) is formed at a rate that is less than 1/1000th of that of the NADPH-containing complex shown here, and it therefore lacks kinetic competence. This finding points up an important catalytic role for NADPH that does not involve its obvious function as a hydride donor and is distinctly separate from this role. In the case of the glutamate dehydrogenase-catalyzed reaction, this "occult role" clearly involves the induction of ketimine formation on the enzyme surface.
D000641:Ammonia / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D007656:Ketoglutaric Acids / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000502:physiology*
D005971:Glutamates; D007656:Ketoglutaric Acids; D018698:Glutamic Acid; D009249:NADP; D000641:Ammonia; D005969:Glutamate Dehydrogenase