Purification and properties of an esterase from Cucurbita maxima fruit tissue

An esterase from the fruit of Cucurbita maxima was purified to apparent homogeneity. The enzyme displays an M_r, of 36 000, a pI value of 4.9, a Stokes radius of 2.70 nm, diffusion coefficient of 9.25 × 10 ^{$ ̄7} cm²/sec, possesses a homogeneous dimeric quaternary structure with a subunit M_r, of 18 000. High esterolytic activity was observed with indophenyl acetate (1510μmol/min/mg protein and p-nitrophenyl acetate (648μmol/min/mg protein) while the enzyme displayed no carboxypeptidase, amidase, proteinase or aminopeptidase activities. Based on indophenyl acetate as substrate, the esterase has an optimum pH of 7.5 to 8.9 and a K_m value of 0. 14 mM at pH 8.0. The esterolytic activity is strongly inhibited by mercaptide-forming and alkylating thiol reagents and by diisopropyl fluorophosphate and phenylmethylsulphonyl fluoride. Product inhibition by indophenyl was competitive (apparent K_i = 90,μM) relative to indophenol acetate and parabolic. Acetate was without effect.