The Mechanism of Action of Methionyl-tRNA Synthetase from Escherichia coli. Inhibition by Adenosine and 8-Aminoadenosine of the Amino-Acid Activation Reaction
In vitro kinetic analysis reveals coupled substrate binding in Escherichia coli methionyl-tRNA synthetase, indicating that amino-acid engagement governs pyrophosphate recruitment.
Key Points
Investigate the catalytic mechanism and active-center coupling for methionyl-adenylate formation in Escherichia coli methionyl-tRNA synthetase using adenosine analogues as competitive inhibitors.
Assayed ATP-PPi exchange reactions catalyzed by methionyl-tRNA synthetase in the presence of adenosine and 8-aminoadenosine inhibitors.
Resolved steady-state kinetic parameters to evaluate interactions between the amino acid, nucleoside inhibitors, and pyrophosphate binding sites.
Methionine markedly elevated the affinity of both adenosine and 8-aminoadenosine for methionyl-tRNA synthetase, confirming cooperative coupling between the amino-acid and nucleoside-binding domains.
Pyrophosphate binding occurred only upon simultaneous occupation of both the amino-acid and nucleoside catalytic sites.
Addition of an amino group at position 8 of the adenine ring substantially increased binding affinity constants for both the nucleoside inhibitor and pyrophosphate.