REENGINEERING THE CATALYTIC LYSINE OF ASPARTATE-AMINOTRANSFERASE BY CHEMICAL ELABORATION OF A GENETICALLY INTRODUCED CYSTEINE

The active-site essential catalytic residue of aspartate aminotransferase, Lys 258, has been converted to Cys (K258C) by site-directed mutagenesis. This mutant retains less than 10(-6) of the wild-type activity with L-aspartate. The deleted general base was functionally replaced by selective (with respect to the other five cysteines in wild type) aminoethylation of the introduced Cys 258 with (2-bromoethyl)amine following reversible protection of the nontarget sulfhydryl groups at different stages of unfolding. The chemically elaborated mutant (K258C-EA) is 10(5) times more reactive than is K258C and has a k(cat) value of approximately 7% of that of wild type (WT). K(m) and K(I) values are similar to those for WT. The acidic pK(a) controlling V/K(Asp) is shifted from 7.3 (WT) to 6.0 (mutant). V/K values for amino acids are approximately 3% of those found for WT, whereas they are approximately 20% for keto acids. The value of D(V) increases from 1.6 for WT to 3.4 for the mutant, indicating that C-alpha proton abstraction constitutes a more significant kinetic barrier for the latter enzyme. A smaller, but still significant, increase in D(V/K(Asp)) from 1.9 in WT to 3.0 in the mutant shows that the forward and reverse commitment factors are inverted by the mutation. The acidic limb of the V/K(Asp) versus pH profile, is lowered by 1.3 pH units, probably reflecting the similar difference in the basicity of the epsilon-NH2 group in gamma-thialysine versus that in lysine. The decreased basicity of this group in K258C-EA is also considered to be principally responsible for the lower values of V(max) and the larger kinetic isotope effects characterizing K258C-EA when compared to wild-type enzyme.