Use of binding energy in catalysis: optimization of rate in a multistep reaction.

The role of binding energy in optimizing the overall rate of catalysis by the tyrosyl-tRNA synthetase from Bacillus stearothermophilus has been investigated by measuring the rate constants for transfer of tyrosine from engineered mutants to tRNA. The residues chosen for mutation are those that were previously identified as binding tyrosyl adenylate and contributing to the rate constant for its formation. It was previously found that tighter binding of the tyrosyl adenylate was accompanied by an increase in the rate constant for its formation. A new linear free energy relationship is presented that links the two. We now find that the rate constant for transfer of Tyr from Tyr-AMP to tRNA decreases with increasing stability of the E.Tyr-AMP complex on mutation of Thr-51. Position 51 is the one that is found to be the most variable of the binding-site residues among the enzymes from different species. The tightness of binding of the intermediate is thus a compromise, since stabilizing the intermediate speeds up the first step but slows down the second. The rate constants for activation and transfer by wild-type enzymes are very similar, which is optimal for the rate of the overall reaction. Those for the mutants diverge so that the rate of overall catalysis is lower.