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

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