SPECIFIC ACTIVATION OF A TYROSINE -] GLYCINE MUTANT OF DELTA-5-3-KETOSTEROID ISOMERASE BY PHENOLS

A key unknown still to be explored concerning the mechanism of Δ5-3ketosteroid isomerase from Pseudomonas testosteroni is the extent of the proton transfer between tyrosine-14 of the enzyme and the C-3 carbonyl oxygen of the steroid substrate. This report is a preliminary study of a system we are developing to allow us eventually to use a Brønsted analysis to measure this transfer. We describe the construction of an expression vector and tyrosine-14 → glycine-14 mutant of the enzyme and its specific activation, in the manner of chemical rescue, by a variety of phenolic compounds. We suggest that the binding region of phenol is very tight and that the level of activation may be a result of steric constraints as well as of differences in the pKa' of the phenol. © 1992 Academic Press, Inc.