MUTANT ASPARTATE-AMINOTRANSFERASE (K258H) WITHOUT PYRIDOXAL-5'-PHOSPHATE-BINDING LYSINE RESIDUE - STRUCTURAL AND CATALYTIC PROPERTIES

If the pyridoxal-phosphate-binding lysine residue 258 of aspartate aminotransferase is exchanged for a histidine residue, the enzyme retains partial catalytic competence [Ziak, M., Jaussi, R., Gehring, H. and Christen, P. (1990) Eur, J. Biochem. 187, 329 333]. The three-dimensional structures of the mutant enzymes of both chicken mitochondria and Escherichia coli were determined at high resolution. The folding patterns of the polypeptide chains proved to be identical to those of the wild-type enzymes, small conformational differences being restricted to parts of the active site. If aspartate or glutamate was added to the pyridoxal form of the mutant enzyme [lambda(max) 392 nm and 330 nm (weak); negative CD at 420 nm, positive CD at 370 nm and 330 nm], the external aldimine (lambda(max) = 430 nm; negative CD at 360 nm and 430 nm) transiently accumulated. Upon addition of 2-oxoglutarate to the pyridoxamine form (lambda(max) 330 nm, positive CD). a putative ketimine intermediate could be detected; however, with oxalacetate, an equilibrium between external aldimine and the pyridoxal form, which was strongly in favour of the former, was established within seconds. The transamination cycle with glutamate and oxalacetate proceeds only three orders of magnitude more slowly than the overall reaction of the wild-type enzyme. The specific activity of the mutant enzyme is 0.1 U/mg at 25-degrees-C and constant from pH 6.0 to 8.5. Reconstitution of the mutant apoenzyme with (4'-H-3]pyridoxamine 5'-phosphate resulted in rapid release of H-3 with a first-order rate constant k' = 5 X 10(-4) s-1 similar to that of the wild-type enzyme. Apparently, in aspartate aminotransferase, histidine can to some extent substitute for the active-site lysine residue. The imidazole ring of H258, however, seems too distant from Calpha and C4' to act efficiently as proton donor/acceptor in the aldimine-ketimine tautomerization. suggesting that the prototropic shift might be mediated by an intervening water molecule. Transimination of the internal to the external aldimine apparently can be replaced by de novo formation of the latter, and by its hydrolysis in the reverse direction.