Kinetic mechanism of chloramphenicol acetyltransferase: the role of ternary complex interconversion in rate determination.

Abstract

Chloramphenicol acetyltransferase (CAT) catalyzes the acetyl-CoA-dependent acetylation of chloramphenicol (Cm) by a ternary complex mechanism and with a random order of addition of substrates. A closer examination of the mechanism of the reaction catalyzed by the type III CAT variant (CATIII) has included the measurement of the individual rate constants by stopped-flow fluorimetry at 5 degrees C. Under all conditions employed, product release from the binary complexes in both forward and reverse reactions was found to be too slow to account for the observed overall rate of turnover for the reaction. Additional, faster routes for product release are achieved via the formation of the nonproductive 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 than kcat (135 s-1 at 5 degrees C), whereas the dissociation rate constants of 3-acetyl-Cm from the ternary complexes with CoA and acetyl-CoA are 120 and 200 s-1, respectively. Arrhenius plots of dissociation rate constants indicate a slow release of products over a broad temperature range. Computer simulations based on the rate constants of CATIII applied to a ternary complex mechanism, assuming random order of substrate addition and product release, yielded nonlinear initial rates of product formation unless both nonproductive ternary complexes were included in the model. Simulated steady-state kinetic analyses based on the latter assumption yielded kinetic parameters that compared favorably with those determined experimentally.(ABSTRACT TRUNCATED AT 250 WORDS)