KINETIC MECHANISM OF CHLORAMPHENICOL ACETYLTRANSFERASE - THE ROLE OF TERNARY COMPLEX INTERCONVERSION IN RATE DETERMINATION

Chloramphenicol acetyltransferase (CAT) catalyzes the acetyl-CoA-depen dent acetylation of chloramphenicol (Cm) by a ternary complex mechanis m and with a random order of addition of substrates. A closer examinat ion of the mechanism of the reaction catalyzed by the type III CAT var iant (CAT(III)) has included the measurement of the individual rate co nstants by stopped-flow fluorimetry at 5 degrees C. Under all conditio ns employed, product release from the binary complexes in both forward and reverse reactions was found to be too slow to account for the obs erved overall rate of turnover for the reaction. Additional, faster ro utes for product release are achieved via the formation of the nonprod uctive ternary complexes (CAT:3-acetyl-Cm:acetyl-CoA and CAT:CoA:Cm). The release of 3-acetyl-Cm from the binary complex is 5-fold slower th an k(cat) (135 s(-1) at 5 degrees C), whereas the dissociation rate co nstants of 3-acetyl-Cm from the ternary complexes with CoA and acetyl- CoA are 120 and 200 s(-1), respectively. Arrhenius plots of dissociati on rate constants indicate a slow release of products over a broad tem perature range. Computer simulations based on the rate constants of CA T(III) applied to a ternary complex mechanism, assuming random order o f substrate addition and product release, yielded nonlinear initial ra tes of product formation unless both nonproductive ternary complexes w ere included in the model. Simulated steady-state kinetic analyses bas ed on the latter assumption yielded kinetic parameters that compared f avorably with those determined experimentally. The proton inventory fo r the reaction catalyzed by CAT(III) is compatible with the involvemen t of proton(s) in one or more rate-determining steps, possibly en rout e td the transition state from the ternary complex of enzyme and subst rates (Cm and acetyl-CoA). Thus, both product release and ternary comp lex interconversion are likely to be involved in rate determination of the CAT(III) reaction.