Site-directed mutagenesis as a probe of enzyme structure and catalysis: tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation

Abstract

Oligodeoxynucleotide-directed mutagenesis was used on the gene of tyrosyl-tRNA synthetase from Bacillus stearothermophilus to produce mutant enzymes altered at the ATP binding site. Deliberate attempts were made to alter rather than destroy enzymic activity so that kinetic measurements may be made to identify the subtle roles of the enzyme-substrate interactions in catalysis. Cys-35, the -SH group of which is involved in binding the 3''-OH of the ribose ring of ATP, was mutated to a serine residue or glycine residue. The mutant enzymes are less active than the wild type and the reduction in activity can be attributed to a decrease in the value of kcat and an increase in Km. The interaction energy of the side chain of Cys-35 with the substrate is not fully realized in the enzyme-substrate complex but is used preferentially to stabilize the transition state. Relative to its absence in the Gly-35 mutant, the side chain of Cys-35 is calculated to stabilize the transition state for PPi exchange by 1.2 kcal/mol and the transition state for aminoacylation by 1.0 kcal/mol.