Effect of substrate concentration, product concentration, and peptides on the in vitro hydrolysis of model ester prodrugs by corneal esterases.

The corneal epithelial fraction of albino rabbits with butyrylcholinesterase-like activity, which was purified by gel filtration chromatography, was studied with respect to its susceptibility to substrate and product inhibition using a pH-stat method. 1- and 2-Naphthyl acetates were used as model ester prodrugs. It was found that the hydrolytic rate of 1- and 2-naphthyl acetates at 1.2 mM was only 53% and 42% of that at 0.06 mM, respectively, suggesting that increasing the dose of an ester prodrug does not necessarily result in a proportional increase in its hydrolytic rate. Product inhibition (by 1- and 2-naphthol) was evident only at a product concentration at least equal to the substrate concentration, suggesting that, at the therapeutic concentrations of an ester prodrug, product inhibition is probably insignificant. Substrate and product inhibition did not appear to occur at the enzyme's active site. Moreover, the neuropeptides enkephalins and their hydrolytic fragments were found to inhibit the hydrolysis of 1-naphthyl acetate, albeit less effectively than compounds such as 1- and 2-naphthol.