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 kcat value of approximately 7% of that of wild type (WT). Km and KI values are similar to those for WT. The acidic pKa controlling V/KAsp 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 DV 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/KAsp) 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/KAsp 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Planas
A
A
Department of Molecular and Cellular Biology, University of California, Berkeley 94720.
Kirsch
J F
JF
D000478:Alkylation; D001219:Aspartate Aminotransferases / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005818:Genetic Engineering; D007700:Kinetics; D008239:Lysine / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed
D001219:Aspartate Aminotransferases; D008239:Lysine; D003545:Cysteine