Functional analysis of Phe-259 and Thr-314 within the substrate binding pocket of the JH esterase of Manduca sexta

Here we analyze the X-ray crystal structure of the JH esterase of the tobacco hornworm Manduca sexta (MsJHE) and identify two non-catalytic amino acid residues, Phe-259 and Thr-314, within the substrate binding pocket that potentially interact with the ester and epoxide, respectively, of JH. The roles of these residues in JH hydrolysis was analyzed by molecular modeling, mutagenesis, and enzyme kinetic characterization using JH III, saturated JH III, and JH III diol as substrates. Saturated JH III was turned over by MsJHE at the same rate as JH III, whereas a Phe-259 to Ile mutant of MsJHE turned over these substrates at roughly 5-fold higher rates. A similar increase in turnover rate was shown by a Thr-314 to Val mutant of MsJHE. These findings suggested that (i) the hydrolysis of the resonance-stabilized ester is not the rate-determining step in the hydrolysis of JH III by JHE as previous believed, and (ii) that interaction between Phe-259 and Thr-314 and the JH acid metabolite reduce the turnover rate.