Mg~(2+)-Free Bacillus 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, nonlinear 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 [Mg~(2+)]_(total). Tryptophan activation by Bacillus stearothermophilus tryptophanyl-tRNA synthetase falls asymptotically to a plateau value 5 orders of magnitude below that observed for the Mg~(2+)-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 × 10~4 attributable to the catalytic effect of Mg~(2+) and an enhanced (K_E~≠ = 1.15 × 10~(-7) M) transition-state binding of Mg~(2+). Factorial analysis indicates that 80% of the reduction in. free energy of activation effected by TrpRS arises from protein-ligand interactions.