Free energy of hydrolysis of tyrosyl adenylate and its binding to wild-type and engineered mutant tyrosyl-tRNA synthetases.
The equilibrium constant for the formation of tyrosyl adenylate and pyrophosphate from ATP and tyrosine in solution has been measured by applying the Haldane relationship to wild-type and three mutant tyrosyl-tRNA synthetases from Bacillus stearothermophilus. The formation constant (=[Tyr-AMP] [PPi]/[ATP] [Tyr]) at pH 7.78, 25 degrees C, and 10 mM MgCl2 is (3.5 +/- 0.5) X 10(-7). This corresponds to a free energy of hydrolysis of tyrosyl adenylate at pH 7.0 and 25 degrees C of -16.7 kcal mol-1. All necessary rate constants had been determined previously for the calculations apart from the dissociation constant of tyrosyl adenylate from its enzyme-bound complex. This was measured by taking advantage of the 100-fold difference in hydrolysis rates of the tyrosyl adenylate when sequestered by the enzyme and when free in solution. These are technically difficult measurements because the dissociation constants are so low and the complexes unstable. The task was simplified by using mutants prepared by site-directed mutagenesis. These were designed to have different rate and equilibrium constants for dissociation of tyrosyl adenylate from the enzyme-bound complexes. The dissociation constants were in the range (3.5-38) X 10(-12) M, with that for wild type at 13 X 10(-12) M. The four enzymes all gave consistent data for the formation constant of tyrosyl adenylate in solution. This not only improves the reliability of the measurement but also provides confirmation of the reliability of the measured kinetic constants for the series of enzymes.
Wells
T N
TN
Ho
C K
CK
Fersht
A R
AR
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D005818:Genetic Engineering; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D008433:Mathematics; D009154:Mutation; D011485:Protein Binding; D013816:Thermodynamics; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism
D000249:Adenosine Monophosphate; D014443:Tyrosine; C019586:tyrosinyl-5'-AMP; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase