Physical and kinetic studies of arginyl transfer ribonucleic acid ligase of Neurospora. A sequential ordered mechanism.

The arginyl-tRNA ligase of Neurosporo has been purified 1,800-fold. The purified preparation catalyzed the esterification of 140 /Imoles of arginine to tRNA per hour per mg of protein at 35”. Polyacrylamide gel electrophoresis revealed a single major band from which the enzyme was estimated to be 95% homogeneous. The apparent molecular weight of the enzyme is about 85,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel titration. No evidence for subunits was obtained. The enzyme required tRNAArg for the ATP-pyrophosphate exchange reaction, and this requirement was not satisfied by periodate-oxidized tRNA. Transfer RNA was also required for the arginine-dependent cleavage of pyrophosphate from ATP. These two reactions (pyrophosphate exchange and pyrophosphate cleavage from ATP) proceed at a faster rate than the esterification reaction. Other differences with the Escherichia coti arginyl-tRNA ligase, including molecular size, amino acid composition, tRNA substrate specificity, and order of substrate addition, were noted. dATP could fully replace ATP in both the pyrophosphate exchange and the esterification reactions. Canavanine could replace arginine, whereas arginine hydroxamate, arginine methyl ester, argininosuccinate, homoarginine, and cy-aminoy-guanidobutyrate were nonreacting competitive inhibitors. The addition of substrates and the release of products were investigated primarily by bisubstrate kinetics, deadend, and product inhibition studies. The kinetic patterns obtained indicated a unique sequential ordered mechanism of substrate addition; tRNA*‘g is bound fist, followed by arginine, and then by ATP. Pyrophosphate and AMP may be released in a random fashion, and arginyl-tRNA is the last product to dissociate from the enzyme. The ligase catalyzed a tRNA-dependent pyrophosphorolysis of exogenous arginyladenylate. The enzyme could also use arginyladenylate as an arginine donor for the esterification of tRNA. The rates of pyrophosphorolysis and esterilication with arginyladenylate were very similar to the ATP-