EXTENT OF PROTON-TRANSFER IN THE TRANSITION-STATES OF THE REACTION CATALYZED BY THE DELTA(5)-3-KETOSTEROID ISOMERASE OF COMAMONAS (PSEUDOMONAS) TESTOSTERONI - SITE-SPECIFIC REPLACEMENT OF THE ACTIVE-SITE BASE, ASPARTATE-38, BY THE WEAKER BASE ALANINE-3-SULFINATE

Previous studies of the mechanism of the steroid isomerase of Comamonas (Pseudomonas) testosteroni have identified aspartate 38 as the proton porter which transfers the substrate's 4 beta proton to the 6 beta position of the product. Consequently, aspartate 38 functions as a base in the deprotonation of the substrate to form a dienol or dienolate intermediate, which then undergoes reprotonation from protonated aspartate 38 at C-6 beta to give the product. We have tried to characterize the transition states for the proton transfers by altering the pK(a)' of aspartate 38 and then determining the effect of the alteration on the kinetics of the enzyme. Alteration of the pK(a)' was accomplished by replacement of the carboxyl carbon of aspartate 38 by sulfur, a change which converts the carboxylate group to the much less basic sulfinate group. Employing Bronsted catalysis theory as applied to the individual steps of the isomerase mechanism, we find that in the enolization step of the reaction proton transfer to aspartate 38 is well advanced in the transition state. In the subsequent ketonization step, proton transfer from aspartate 38 has barely started when that transition state is reached. A series of mutant KSIs with alternative bases at position 38 have been constructed using a combination of site-directed mutagenesis and chemical modification: Asp-38 to Glu (D38E), His (D38H), and S-(carboxymethyl)cysteine (D38CMC). While the D38H and D38E mutants both retain significant isomerase activity, D38CMC is essentially inert. From the results of kinetic experiments it is possible to get a qualitative idea of the sensitivity of the enzyme's catalytic ability to the location of the base responsible for proton transfer.