Lack of Evidence for a Tetrahedral Intermediate in the Hydrolysis of Nitroanilide Substrates by Serine Proteinases

Abstract

A stopped-flow apparatus was used to reinvestigate reports, based on the observation of ''burst'' kinetics, of an intermediate prior to the acyl-enzyme complex in hydrolysis reactions of anilides catalyzed by trypsin and elastase. The hydrolysis of several anilide substrates by bovine and porcine trypsin (EC 3.4.21.4) and porcine elastase (EC 3.4.21.11) was studied between -30 and +20.degree. C. In no case was true ''burst'' kinetics recorded. Confusing spectral changes can arise from incomplete mixing, thermal gradients or heterogeneity of the substrate. There is no solid spectroscopic evidence at present for the existence of a tetrahedral intermediate in the hydrolysis of amides by serine proteinases. The substrate N.sbd.acetyl.sbd.L.sbd.alanyl.sbd.L-prolyl.sbd.L.sbd.alanine 4-nitroanilide is a mixture of 2 isomers trans and cis about the L.sbd.alanyl.sbd.L.sbd.propyl peptide bond. It appears that elastase hydrolyzes the cis isomer more rapidly than the trans isomer, and this could lead to false ''burst'' kinetics. A stopped-flow apparatus with novel features, designed for cryoenzymology and used for this work and that is adaptable to a variety of spectrophotometers is described. Solutions can be handled under anaerobic conditions. A window allows the drive syringes to be observed or exposed to light for photochemical experiments. The apparatus operates over the temperature range -35 to +25.degree. C. The dead time is < 5 ms. A recording system is described that permits the following of reactions over a wide time scale covering half-times of the order of several ms to hours.