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.
Weinreb
Violetta
V
Department of Biochemistry and Biophysics, CB 7260, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
Carter
Charles W
CW
D000255:Adenosine Triphosphate / Q000737:chemistry; D001671:Biochemistry / Q000379:methods; D002384:Catalysis; D018360:Crystallography, X-Ray; D004492:Edetic Acid / Q000737:chemistry; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000378:metabolism; D007477:Ions; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000737:chemistry*; D008956:Models, Chemical; D010766:Phosphorylation; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry*; D014369:Tryptophan-tRNA Ligase / Q000737:chemistry*
D007477:Ions; D008024:Ligands; D014364:Tryptophan; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D014369:Tryptophan-tRNA Ligase; D008274:Magnesium