HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nazario, M. Articles by Evans, J. A. Search for Related Content PubMed PubMed Citation Articles by Nazario, M. Articles by Evans, J. A. Social Bookmarking                      What's this?

Physical and Kinetic Studies of Arginyl Transfer Ribonucleic Acid Ligase of Neurospora

A SEQUENTIAL ORDERED MECHANISM

From the ¹ From the Department of Biochemistry and Molecular Biology, University of Kansas Medical School, Kansas City, Kansas 66103

The arginyl-tRNA ligase of Neurospora has been purified 1,800-fold. The purified preparation catalyzed the esterification of 140 µmoles of arginine to tRNA per hour per mg of protein of 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 filtration. No evidence for subunits was obtained.

The enzyme required tRNA^Arg 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 coli 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 -amino--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^Arg is bound first, 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 esterification with arginyladenylate were very similar to the ATPpyrophosphate exchange and over-all esterification rates, respectively.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				L.-T. Guo, X.-L. Chen, B.-T. Zhao, Y. Shi, W. Li, H. Xue, and Y.-X. Jin Human tryptophanyl-tRNA synthetase is switched to a tRNA-dependent mode for tryptophan activation by mutations at V85 and I311 Nucleic Acids Res., September 27, 2007; 35(17): 5934 - 5943. [Abstract] [Full Text] [PDF]