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

We have used a stopped‐flow apparatus to reinvestigate reports, based on the observation of ‘burst’ kinetics, of an intermediateprior to the acyl‐enzyme complex in hydrolyis reactions of anilides catalyzed by trypsin and elastase [M.W. Hunkapiller, M.D. Forgac and J.H. Richards (1976) Biochemistry 15 , 5581–5588; D.D. Petkov (1978) Biochim. Biophys. Acta, 523 , 538–541; A.L. Fink and P. Meehan (1979) Proc. Natl Acad. Sci. USA, 76 , 1566–1569; P. Compton and A.L. Fink (1980) Biochem. Biophys. Res. Commun. 93 , 427–431]. We studied the hydrolysis of seferal anilide substrates by bovien and porcine elastase between ‐30°C and 20°C. In no case did we record true ‘burst’ kinetics. We show that confusing spectral changes can arise from incomplete mixing, thermal gradients, or heterogeneity of the substrate. We conclude that 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 ‐acetyl‐ l ‐alanyl‐ l ‐prolyl‐ l ‐alanine 4‐nitroanilide is a mixture of two isomers trans and cis about the l ‐alanyl‐ l ‐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. We describe the construction of the stopped‐flow apparatus designed for cryoenzymology used for this work that has novel features and is adaptable to a variety of spetrophotometers. Solutions can be handled under anaerobic conditions. A window allows teh drive syringes to be observed or exposed to light for photochemical experiments. The apparatus operates over the temperature range −35°C to +25°C. The dead time is under 5 ms. A recording system is described that permits one to follow reations over a wide time scale covering half‐times of the order of several milliseconds to hours.