Site-directed mutagenesis as a probe of enzyme structure and catalysis: tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation.
Oligodeoxynucleotide-directed mutagenesis has been used on the gene of tyrosyl-tRNA synthetase from Bacillus stearothermophilus to produce mutant enzymes altered at the adenosine 5'-triphosphate (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, has been mutated to a serine residue [Winter, G., Fersht, A. R., Wilkinson, A. J., Zoller, M., & Smith, M. (1982) Nature (London) 299, 756-758] 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. Thus, 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 pyrophosphate exchange by 1.2 kcal/mol and the transition state for aminoacylation by 1.0 kcal/mol.
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D001483:Base Sequence; D003545:Cysteine; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D005998:Glycine; D007700:Kinetics; D009154:Mutation; D014448:Tyrosine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism
D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase; D003545:Cysteine; D005998:Glycine