Title: Studies on the Mechanism of Action of Aspartate Aminotransferase (AspAT) by Cryoenzymological and Isotope Techniques
Abstract: SummaryCryoenzymological tools were applied to render enzyme- substrate intermediates of AspAT accessible to kinetic, chemical, and structural analysis. Starting from the pyridoxal or pyridoxamine form of AspAT, single half-reactions of enzymic transamination could be monitored at temperatures below −40°C with the amino acid substrates glutamate, aspartate and cysteine sulfinate. The cryosolvent has no significant effects on the catalytic and structural properties of the enzyme as indicated by virtually unchanged kinetic parameters and a linear Arrhenius plot. Below −60°C the formation of stable intermediates could be followed.In order to understand initial events occurring in the reverse half-reaction of transamination, we investigated the acidity of pyridoxamine 5′-phosphate (PMP) at the pro-S position of C-4′ in its active site environment in the absence of an oxo acid substrate, i.e. independently of the formation of the ketimine intermediate. Reconstitution of mitochondrial apoAspAT with [4′-3H] PMP resulted in a stereospecific exchange of pro-S 3H with solvent. Experiments with various ligands indicate that exchange preferably occurs when the open/closed conformational equilibrium of the enzyme is shifted toward the closed conformation.
Publication Year: 1987
Publication Date: 1987-01-01
Language: en
Type: book-chapter
Indexed In: ['crossref']
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Cited By Count: 1
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