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Science 17 March 1989:
Vol. 243. no. 4897, pp. 1485 - 1488
DOI: 10.1126/science.2538921
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Articles

Science, Vol 243, Issue 4897, 1485-1488
Copyright © 1989 by American Association for the Advancement of Science

articles

Direct Bronsted analysis of the restoration of activity to a mutant enzyme by exogenous amines

MD Toney and JF Kirsch

Department of Biochemistry, University of California, Berkeley 94720.

A true Bronsted analysis of proton transfer in an enzyme mechanism is made possible by the chemical rescue of an inactive mutant of aspartate aminotransferase, where the endogenous general base, Lys258, is replaced with Ala by site-directed mutagenesis. Catalytic activity is restored to this inactive mutant by exogenous amines. The eleven amines studied generate a Bronsted correlation with beta of 0.4 for the transamination of cysteine sulfinate, when steric effects are included in the regression analysis. Localized mutagenesis thus allows the classical Bronsted analysis of transition-state structure to be applied to enzyme-catalyzed reactions.


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