Reconstruction by site-directed mutagenesis of the transition state for the activation of tyrosine by the tyrosyl-tRNA synthetase: a mobile loop envelopes the transition state in an induced-fit mechanism.
Site-directed mutagenesis of the tyrosyl-tRNA synthetase followed by kinetic studies has shown that residues which are distant from the active site of the free enzyme are brought into play as the structure of the enzyme changes during catalysis. Positively charged side chains which are in mobile loops of the enzyme envelope the negatively charged pyrophosphate moiety during the transition state for the formation of tyrosyl adenylate in an induced-fit mechanism. Residues Lys-82 and Arg-86, which are on one side of the rim of the binding site pocket, and Lys-230 and Lys-233, which are on the other side, have been mutated to alanine residues and also to asparagine or glutamine. The resultant mutants still form 1 mol of tyrosyl adenylate/mol of dimer but with rate constants up to 8000 times lower. Construction of difference energy diagrams reveals that all the residues specifically interact with the transition state for the reaction and with pyrophosphate in the E.Tyr-AMP.PPi complex. Yet, the epsilon-NH3+ groups of Lys-230 and Lys-233 in the crystalline enzyme are at least 8 A too far away to interact with the pyrophosphate moiety in the transition state at the same time as do Lys-82 and Arg-86. Binding of substrates must, therefore, induce a conformational change in the enzyme that brings these residues into range. Consistent with this proposal is the observation that all four residues are in flexible regions of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Fersht
A R
AR
Department of Chemistry, Imperial College of Science and Technology, London, U.K.
Knill-Jones
J W
JW
Bedouelle
H
H
Winter
G
G
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008433:Mathematics; D009154:Mutation; D011487:Protein Conformation; D013816:Thermodynamics; D046249:Transfer RNA Aminoacylation; D014443:Tyrosine / Q000378:metabolism*; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*
D014443:Tyrosine; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase