FUNCTIONAL-ROLE OF THE AMINO-TERMINAL MOBILE SEGMENT IN CATALYSIS BY PORCINE CYTOSOLIC ASPARTATE-AMINOTRANSFERASE - CRITICAL IMPORTANCE OF VAL(17) AND PHE(18) FOR PRODUCTIVE BINDING OF SUBSTRATES

A notable feature of porcine cytosolic aspartate aminotransferase is t he closure of the active site cleft by a mobile amino-terminal segment (residues 15-40) upon binding substrate. The functional roles of Va1( 17) and Phe(18), residues that are part of the mobile loop, have been studied in the site-directed mutants in which the size and hydrophobic nature of these residues have been changed. Absorption, circular dich roism spectra, susceptibility to protease 401, and thermal stability d id not differ appreciably between wild type and mutant enzymes. In the overall transamination between aspartate and 2-oxoglutarate, V17A rep resented a typical K-m mutant while V17I retained the substrate bindin g affinity fairly well. In contrast, replacement of Phe(18) by Ala res ulted in a large decrease in both catalytic rate and binding affinity for substrates. F18W, F18Y, and F18H showed a moderate decrease in k(c at) and a considerable increase in K-m values. Single-turnover reactio ns with four individual substrates yielded analogous results to those obtained for the overall reaction and, in addition, revealed that k/K- d values of mutants F18A and F18H were over 10 times lower for C-5 sub strates (glutamate and 2-oxoglutarate) than those for C-4 substrates ( aspartate and oxalacetate). All mutant enzymes showed variously increa sed K-d values for substrate analogs such as 2-methylaspartate, succin ate, and glutarate. H-1 MMR observations of F18H, in which His(18) ser ved as a built-in probe, were in accord with the behavior that would b e expected from the conformational transition. We conclude that, altho ugh Val(17) and Phe(18) may not be essential for catalysis, the presen ce of a bulky residue of appropriate size at each position is critical for productive binding of substrate.