Trichodiene synthase: Synthesis and inhibition kinetics of 12-fluoro-farnesylphosphonophosphate for sesquiterpene cyclases

Abstract

trans, trans-Farnesyl diphosphate (FPP) serves as a universal substrate for a large family of sesquiterpene cyclases that are responsible for biosynthesis of more than 300 structurally diverse sesquiterpenes in nature. A new FPP substrate analogue, 12-fluoro-farnesylphosphonophosphate (12-F-F-CH2PP), was synthesized in this paper for applications on kinetic and mechanistic studies of the enzyme family. Trichodiene synthase (TS), a sesquiterpene cyclase, catalyzes the conversion of trans,transfarnesyl diphosphate (FPP) to trichodiene. 12-F-F-CH2PP was tested as a potential inhibitor of TS. Inactivation and inhibition kinetic experiments showed that 12-F-F-CH2PP was not a mechanism-based inactivator for TS; instead, a mixed-type reversible inhibition was observed with inhibition constants K-i1 = 2.33 +/- 0.50 mu M and K-i2 25.80 +/- 7.70 mu M, values close to those previously determined for farnesylphosphonophosphate, K-i1 3.25 mu M and K-i2 = 9-10 mu M Although 12-F-F-CH2PP did not irreversibly inactivate TS, this new analogue serves as a potential active-site directed inactivator and mechanistic probe of other sesquiterpene cyclases and FPP-utilizing enzymes, which utilize FPP as a common acyclic substrate.