文章基本信息

标题：Detection and accumulation of tetrahedral intermediates in elastase catalysis
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作者：A L Fink ; P Meehan
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1979
卷号：76
期号：4
页码：1566-1569
DOI：10.1073/pnas.76.4.1566
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Tetrahedral intermediates in the reaction of elastase with specific di- and tripeptide p-nitroanilide substrates have been detected, accumulated, and stabilized at high pH by using subzero temperatures and fluid aqueous/organic cryosolvents. The tetrahedral adducts are characterized by spectra with lambda max of 359 +/- 2 nm, compared with thata of 380 nm for p-nitroaniline and 315-320 nm for the substrates. The maximal concentration of intermediate that could be accumulated varied with the different substrates from 40 to 100% of the active enzyme present. The pH dependence of the reactions indicated that formation of the tetrahedral intermediates was rate-limiting at low pH (pK* = 7.0 at -39 degrees C) and that collapse to the acylenzymes was rate-determining at high pH. When corrected for the effect of temperature and cosolvent, the rate of intermediate formation was in good agreement with that measured at 25 degrees C in aqueous solution by stopped-flow techniques.