Kinetic and thermodynamic properties of wild-type and engineered mutants of tyrosyl-tRNA synthetase analyzed by pyrophosphate-exchange kinetics.
The first step of the reaction catalyzed by the aminoacyl-tRNA synthetases is the formation of enzyme-bound aminoacyl adenylate. The steady-state kinetics of this step has conventionally been studied by measuring the rate of isotopic exchange between pyrophosphate and ATP. A simple kinetic analysis of the pyrophosphate-exchange reaction catalyzed by the tyrosyl-tRNA synthetase from Bacillus stearothermophilus is given in which all the observed rate and binding constants can be assigned to identifiable physical processes under a variety of limiting conditions. The free energies of binding to the enzyme of tyrosine, ATP, and the transition state for tyrosyl adenylate formation can be measured in relatively straightforward experiments. The excellent agreement between parameters measured in these experiments and those from earlier pre-steady-state kinetics confirms that the intermediates isolated in the presteady state are kinetically competent. The dissociation constant of ATP from the unligated enzyme, a constant that has previously been experimentally inaccessible, has been measured for wild-type and several mutant enzymes. The changes in enthalpy and entropy of activation on mutation have been measured by a rapid procedure for mutants that have altered contacts with tyrosine and ATP. Those mutants that have large changes of enthalpy and entropy of binding are likely to have structural changes and so warrant further examination by protein crystallography.
Wells
T N
TN
MRC Unit for Protein Function and Design, University Chemical Laboratory, Cambridge, U.K.
Knill-Jones
J W
JW
Gray
T E
TE
Fersht
A R
AR
D000255:Adenosine Triphosphate / Q000378:metabolism; D004252:DNA Mutational Analysis; D011756:Diphosphates / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D007700:Kinetics; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014443:Tyrosine / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*
D011756:Diphosphates; D011994:Recombinant Proteins; D014443:Tyrosine; D000255:Adenosine Triphosphate; D014448:Tyrosine-tRNA Ligase