Dissection of the structure and activity of the tyrosyl-tRNA synthetase by site-directed mutagenesis.
To understand an enzyme reaction, one has to characterize the bound substrates, intermediates, products, and transition states on the reaction pathway and determine the interaction energies between them and the enzyme as the reaction proceeds. Site-directed mutagenesis is invaluable in this task, enabling the systematic dissection of the active site. Residues involved in catalysis may be detected and the energetics probed. The contributions of each hydrogen-bonding site chain in the active site of the tyrosyl-tRNA synthetase to binding and catalysis are being determined by making sensible mutations, which remove defined interactions with the substrates. The difference in free energy between complexes of wild-type and mutant enzymes gives the apparent binding energy of the relevant side chain in each complex. By this means, the following have been determined: the contributions of different types of hydrogen bonds to specificity; their roles in catalysis; the importance of enzyme--substrate versus enzyme--transition-state versus enzyme--intermediate complementarity; the fine tuning of enzyme catalysis during "evolution"; and the existence of linear free energy relationships between rate and binding constants. Prior to these studies, the mechanism of activation by the aminoacyl-tRNA synthetases was totally unknown. It is now seen that catalysis results solely from the use of binding energy. There are residues that do not bind the substrates in the ground state but stabilize just the transition state, consistent with the classical ideas of Haldane and Pauling of enzyme--transition-state complementarity. There are, however, regions of the protein that bind the ribose ring more tightly in the intermediate tyrosyl adenylate than in the transition state.(ABSTRACT TRUNCATED AT 250 WORDS)
Fersht
A R
AR
Department of Chemistry, Imperial College of Science and Technology, London, U.K.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006860:Hydrogen Bonding; D009154:Mutation; D011485:Protein Binding; D012343:RNA, Transfer / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*
D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase