Protection of an unstable reaction intermediate examined with linear free energy relationships in tyrosyl-tRNA synthetase

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Abstract
Linear free energy relationships (LFERs) are powerful tools in the search to understand the relationship between molecular structure and activity. They frequently link the changes in the rate constants caused by alterations in structure. In physical-organic chemistry, these have been interpreted to give information on the structure of the transition state. Similar phenomena have been observed for reactions catalyzed by a series of engineered mutants of tyrosyl-tRNA synthetase from Bacillus stearothermophilus. LFERs are applied in this study to probe how the enzyme minimizes its side reactions. A linear free energy relationship is shown between the binding of the unstable enzyme-tyrosyl adenylate complex and its rate constant of hydrolysis. However, mutations of a key residue, His48, show significant deviation from the relationship, implying a role for the side chain in protection of the complex from hydroxide attack. A second linear free energy relationship is shown linking the rate and equilibrium constants for tyrosyl adenylate binding to the enzyme. Four distinct classes of mutation are discussed in the context of this relationship. The data from all but one of these groups of mutations conform well to linear free energy relationship between the disociation rate and dissociation equilibrium constants for the enzyme-tyrosyl adenylate complex with slope .beta. = 1.01 .+-. 0.08. The specificity of binding of tyrosyl adenylate is determined solely by its dissociation rate constant of the intermediate, and the mutations have relatively little effect on the enzyme-tyrosyl adenylate association rate. A detailed discussion of the methods of analyzing the statistical significance of linear free energy relationships and a guideline for the identification of outliers are given.