Transfer of C alpha-hydrogen of glutamate to coenzyme of aspartate aminotransferase during transamination reaction.
During the transamination reaction of mitochondrial aspartate aminotransferase, transfer of tritium from the alpha-position of glutamate to the pro-S position of C4' of pyridoxamine 5'-phosphate was detected. A fast mixing and quenching device had to be used in order to reduce the number of transamination cycles undergone by the enzyme and thus to minimize the accompanying exchange of label with water. The extent of transfer of label (mean value 1.5%; range 0.8-4%) indicates that the 1,3-prototropic shift follows a stepwise rather than a concerted mechanism and that a single acid/base group is responsible for the proton transfer. The actual extent of proton transfer has to be much higher because the rate of alpha-tritium exchange with solvent was only approximately 10% of that of the turnover of unlabeled substrate, reflecting either an isotope effect or a retention of the tritium label in the reaction center during tautomerization. Under the assumption of an isotope effect, the actual transfer may be estimated to be 13%. This value is consistent with the notion of Lys-258 acting as the proton transferring group in which case the maximal value of transfer in an active site not accessible to solvent during the 1,3-prototropic shift would be 33%. However, alternative mechanisms involving Tyr-70 or a water molecule enclosed in the active site serving as acid/base group cannot be excluded on the basis of the present results. Furthermore, in these investigations aspartate aminotransferase was found to catalyze also the exchange of tritium from the beta-position of glutamate, though at a rate 350 times slower than that of the alpha-exchange.
D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D003067:Coenzymes / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D066298:In Vitro Techniques; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D011733:Pyridoxamine / Q000031:analogs & derivatives; D014316:Tritium
D003067:Coenzymes; D005971:Glutamates; D014316:Tritium; D018698:Glutamic Acid; D011733:Pyridoxamine; D001219:Aspartate Aminotransferases; C010627:pyridoxamine phosphate