3-octylthio-1,1,1-trifluoro-2-propanone, a high affinity and slow binding inhibitor of juvenile hormone esterase from Trichoplusia ni (hübner)

The nature of the inhibition of juvenile hormone esterase (JHE) from Trichoplusia ni (T. ni) by 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP) was studied and the kinetic data presented are consistent with the mechanism of a slow and tight binding inhibitor. With the use of the above inhibitor, it was demonstrated that the classical methods, i.e. Lineweaver and Burk, which are based on the assumptions of a steady state are inadequate for determining the mechanism and the inhibition constants for the reaction of OTFP with JHE. The reaction was shown to be reversible based on the non-steady state kinetics with a dissocation constant (K_i) of ∼ 1.2 × 10^-10 M. Values of 3.37 × 10⁷ M^-1 min^-1 and 4.1 × 10^-3 min^-1 for the forward and reverse rate constants were calculated and indicate the nature of slow binding inhibition in comparison to ordinary substrate reactions. An application of Ackermann-Potter plots of tight binding inhibition was presented, and their usefulness in measuring the enzyme molar equivalency and its catalytic number towards JH-I and JH-III was evaluated. The molar equivalency of JHE in T. ni haemolymph which hydrolyzes JH-III (at a final concentration of 5.0 × 10^-6 M) at a rate of 33.8 nmol/min-ml was found to be 1.6 × 10^-6 M. The catalytic numbers for JH-I and JH-III were 37.1 and 19.4 min^-1, respectively. These new kinetic parameters, in addition to the Michaelis constant (K_m) and the maximum velocity (V_max) were applied to a study of enzyme-substrate specificity and an evaluation of the role of JHE in the regulation of JH-titre.