Pre-steady-state kinetic analysis of the trichodiene synthase reaction pathway.

Abstract

The pre-steady-state kinetics of the trichodiene synthase reaction were investigated by rapid chemical quench methods. The single-turnover rate was found to be 3.5-3.8 s-1, a rate 40 times faster than the steady-state catalytic rate (kcat = 0.09 s-1) for trichodiene synthase-catalyzed conversion of farnesyl diphosphate (FPP) to trichodiene at 15 degrees C. In a multiturnover experiment, a burst phase (kb = 4.2 s-1) corresponding to the accumulation of trichodiene on the surface of the enzyme was followed by a slower, steady-state release of products (klin = 0.086 s-1) which corresponds to kcat. These results strongly suggest that the release of trichodiene from the enzyme active site is the rate-limiting step in the overall reaction, while the consumption of FPP is the step which limits chemical catalysis at the active site. Single-turnover experiments with trichodiene synthase mutant D101E, for which the steady-state rate constant kcat is 1/3 of that of wild type, revealed that the mutation actually depresses the rate of FPP consumption by a factor of 100. The deuterium isotope effect on the consumption of [1-2H,1,2-14C]FPP was found to be 1.11 +/- 0.06. Single turnover reactions of [1,2-14C]FPP catalyzed by trichodiene synthase were carried out at 4, 15, or 30 degrees C in an effort to provide direct observation of the proposed intermediate nerolidyl diphosphate (NPP). However, no NPP was detected, indicating that the conversion of NPP must be too fast to be observed within the detection limits of the assay. Taken together, these observations suggest that the isomerization of FPP to NPP is the step which limits the rate of chemical catalysis in the trichodiene synthase reaction pathway.