AbstractDifluoro-oxaloacetate reacts with the aminic form of aspartate transaminase, forming a tight reversible complex as shown in steady state inhibition studies. Spectrophotmetric evidence indicates slow transamination of the analogue and allowed tentative identification of some of the reaction intermediates, together with the rate constants for their formation and removal. Difluoro-oxaloacetate forms an abortive complex with the aldimine form of transaminase giving rise to observable changes in the 19F n.m.r. signal of the ligand. The pH dependences of line width and chemical shift show inflexion points at pH 5.4 and ph 8.5 implicating groups with pKs in these regions in binding dicarboxylate ligands to the active site. With the apoenzyme, the pH profile of the n.m.r. parameters lacks the inflexion point at pH 5.4, providing evidence that the Schiff-base nitrogen (pK 5.4) is one of the carboxylate binding sites. The direction of n.m.r. shifts implies a non-polar microenvironment at the active site. Steady state kinetics of the NADH reduction of dif1uoro-oxa1oacetate catalysed by malate dehydrogenase show that the fluoro-analogue is a good substrate for the enzyme and follows the same kinetic mechanism as that of oxaloacetate. Binding of the analogue both directly to the enzyme and in a ternary abortive complex was shown in protection experiments using iodoacetamide. The presence of the co-enzyme enhanced the affinity of the enzyme for the keto acid. The 19F n.m.r. signal of difluoro-oxaloacetate shifts downfield and broadens in the presence of malate dehydrogenase and NAD. Difluoro-oxaloacetate is an effective inhibitor of malate dehydrogenase, the inhibition is complex, and appears to result from the formation of abortive binary and ternary complexes, together with alternate product Inhibition. DL-Difluoromalate was found not to be a substrate for malate dehydrogenase, but was a pure non-competitive inhibitor of difluoro-oxaloacetate reduction catalysed by malate dehydrogenase.
|Date of Award||1978|
Studies on the active sites and mechanisms of aspartate transaminase and malate dehydrogenase.
Smith, G. D. (Author). 1978
Student thesis: Doctoral Thesis › PhD