Mg2+-free B. stearothermophilus Tryptophanyl-tRNA Synthetase Retains a Major Fraction of the Overall Rate Enhancement for Tryptophan Activation

摘要

Few experimental data are available for rates of enzymatic phosphoryl-transfer reactions in the absence of the divalent metal ions associated with such reactions. Such data are of interest for amino acid activation by class Ic aminoacyl-tRNA synthetases, for which there is substantial evidence that binding energy of ATP may account for a major fraction of the overall rate enhancement, and it is crucial to know if these effects themselves depend on the divalent metal ion. We describe a nested, non-linear model for the sum of metal-free and metal-catalyzed activities and its use in determining metal-free enzyme activity jointly with transition-state metal binding affinity, by fitting observed values obtained from Mg²⁺-depleted assays with increasing [EDTA] at known [Mg²⁺]total. Tryptophan activation by B. stearothermophilus tryptophanyl-tRNA synthetase falls asymptotically to a plateau value five orders of magnitude below that observed for the Mg²⁺-supplemented enzyme at EDTA concentrations that reduce the free metal concentration to <1 pmolar. The fitted regression model parameters yield a relative rate acceleration of 9.3 × 10⁴ attributable to the catalytic effect of Mg²⁺ and an enhanced (KE^‡ = 1.15 × 10⁻⁷ M) transition-state binding of Mg²⁺. Factorial analysis indicates that 80% of the reduction in free energy of activation effected by TrpRS arises from protein-ligand interactions.