INSIGHTS INTO THE CATALYTIC MECHANISM AND ACTIVE-SITE ENVIRONMENT OF COMAMONAS-TESTOSTERONI DELTA(5)-3-KETOSTEROID ISOMERASE AS REVEALED BYSITE-DIRECTED MUTAGENESIS OF THE CATALYTIC BASE ASPARTATE-38

Delta(5)-3-Ketosteroid isomerase (KSI) of Comamonas testosteroni catal yzes the isomerization of a wide variety of Delta(5(6)) and Delta(5(10 )) steroids through the formation of an enzyme bound dienol(ate) inter mediate. Asp-38 has been strongly implicated in catalysis, apparently serving as a proton shuttle. In this paper the results of a detailed k inetic characterization of the KSI mutants D38E and D38H are presented . Both mutants retain significant activity, with k(cat) and k(cat)/K-m values 10(3)-10(4) times greater than the D38N mutant. The results al low for a qualitative assessment of the sensitivity of the enzymes cat alytic capability to the positioning and chemical nature of the cataly tic base. The near identity of the ratios of k(cat)-5-AND/k(cat)(5,10- EST) is most easily explained by a mechanism in which the second chemi cal step, reketonization of the intermediate dienol(ate), is not signi ficantly rate determining. The pH dependence of the rate constants for the D38E and D38H mutants is found to be consistent with earlier prop osals that an as yet unidentified titrating functional group is presen t in the active site and indicates that the electrostatic environment of residue 38 is hydrophobic and positively charged.