Specificity of the Valyl Ribonucleic Acid Synthetase from Escherichia coli in the Binding of Valine Analogues

Abstract

Abstract Using the valyl-tRNA synthetase from Escherichia coli Km, Ki, and Vmax values have been determined for a variety of potential amino acid substrates and structural analogues of these potential substrates using the ATP:32PPi exchange reaction. Using these kinetic constants, comparisons have been made concerning (a) the relative binding affinity of the enzyme for amino acids and their analogues in the initial binding or recognition reaction, and (b) the relative effectiveness of these same amino acids and analogues in supporting the exchange reaction. It was found that the enzyme had a broader specificity in the initial binding reaction than in the carboxyl activation reaction. Notably, the stereospecificity in the activation reaction appeared to be absolute while it was considerably less than absolute in the binding reaction. The degree of stereospecificity in the binding reaction appeared to be related to the size and structure of the aliphatic alkyl group of the bound amino acid. In the case of valine, the binding affinity was about 50 times greater for the l isomer than for the d isomer; however, in the cases of alanine and α-aminobutyric acid, where the aliphatic alkyl group is smaller than that of valine, there appeared to be a preference for the d isomer in the binding reaction. The presence of an α-carboxyl group in the molecule was not obligatory for effective binding; however, in a number of instances, the presence of the α-carboxyl group of potential substrates and competitive inhibitors appeared to influence the specificity of the enzyme by diminishing the affinity between the enzyme and the bound molecule.