Beef pancreas tryptophanyl-tRNA synthetase. Order of substrate binding in ATP-( 32P)-pyrophosphate exchange reaction

Abstract

Summary The order of substrate binding to tryptophanyl-tRNA synthetase purified from beef pancreas was studied. Kinetics of 32 P PP i -ATP isotope exchange reaction catalyzed by the enzyme were measured in a concentration range of each substrate and also in the presence of various concentrations of a substrate analog, tryptamine. The set of data obtained was treated by means of so called «sequential planning of experiment which allows one to extract fully the information from experimental kinetic data. Three possible orders of substrate binding were considered, namely: (1) ATP first, tryptophan second — (2) tryptophan first, ATP second — (3) random order. Among these sequences the probability of the first one (ATP first, tryptophan second) was found to be close to 100 p. cent whereas two thers were close to zero probability. The high degree of discrimination between various mechanisms was achieved only in the case where all the kinetic data obtained, i.e. both in the presence and absence of the inhibitor, were taken into account in the treatment of the set of experimental points. The discriminatory ability of the method developed is insufficient if the experimental points obtained in the presence of inhibitor are neglected. The procedure developed for the treatment of kinetic data is versatile and can be used for the determination of the sequence of substrate binding for any purified aminoacyl-tRNA synthetase. Any other hypothetical mechanism for the isotope exchange reaction catalyzed by the same enzyme could be treated in a similar manner. The established order of substrate binding should not be extrapolated a priori to other synthetases and to the same enzyme in the presence of tRNA.