Mg2+-free Bacillus stearothermophilus tryptophanyl-tRNA synthetase retains a major fraction of the overall rate enhancement for tryptophan activation.

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 [Mg2+]total. Tryptophan activation by Bacillus stearothermophilus tryptophanyl-tRNA synthetase falls asymptotically to a plateau value 5 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 x 10(4) attributable to the catalytic effect of Mg2+ and an enhanced (K(E)(double dagger) = 1.15 x 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.