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 delta 5-3-ketosteroid 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.