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Science 22 April 1988:
Vol. 240. no. 4851, pp. 521 - 523
DOI: 10.1126/science.3282306
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Articles

Science, Vol 240, Issue 4851, 521-523
Copyright © 1988 by American Association for the Advancement of Science

articles

Evidence from cassette mutagenesis for a structure-function motif in a protein of unknown structure

ND Clarke, DC Lien, and P Schimmel

Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.

The three-dimensional structure of most enzymes is unknown; however, many enzymes may have structural motifs similar to those in the known structures of functionally related enzymes. Evidence is presented that an enzyme of unknown structure [Ile-transfer RNA (tRNA) synthetase] may share a functionally important structural motif with an enzyme of related function (Tyr-tRNA synthetase). This approach involves (i) identifying segments of Ile-tRNA synthetase that have been unusually conserved during evolution, (ii) predicting the function of one such segment by assuming a structural relation between Ile-tRNA synthetase and Tyr-tRNA synthetase, and (iii) testing the predicted function by mutagenesis and subsequent biochemical analysis. Random mutations were introduced by cassette mutagenesis into a ten-amino-acid segment of Ile-tRNA synthetase that was predicted to be involved in the formation of the binding site for isoleucine. Few amino acid substitutions appear to be tolerated in this region. However, one substitution (independently isolated twice) increased the Michaelis constant Km for isoleucine in the adenylate synthesis reaction by greater than 6000-fold, but had little effect on the Km for adenosine triphosphate, the apparent Km for tRNA, or the rate constant kcat.


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Generation of Dominant Selectable Markers for Resistance to Pseudomonic Acid by Cloning and Mutagenesis of the ileS Gene from the Archaeon Methanosarcina barkeri Fusaro.
P. Boccazzi, J. K. Zhang, and W. W. Metcalf (2000)
J. Bacteriol. 182, 2611-2618
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