弹性蛋白酶催化过程中四面体中间体的检测与积累
Detection and accumulation of tetrahedral intermediates in elastase catalysis.
作者信息
Fink A L, Meehan P
出版信息
Proc Natl Acad Sci U S A. 1979 Apr;76(4):1566-9. doi: 10.1073/pnas.76.4.1566.
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.
通过使用零下温度和流体水/有机冷冻溶剂，在高pH值下检测、积累并稳定了弹性蛋白酶与特定二肽和三肽对硝基苯胺底物反应中的四面体中间体。四面体加合物的特征光谱的最大波长为359±2 nm，相比之下，对硝基苯胺的为380 nm，底物的为315 - 320 nm。可积累的中间体的最大浓度因不同底物而异，为存在的活性酶的40%至100%。反应的pH依赖性表明，四面体中间体的形成在低pH值下是限速步骤（在-39℃时pK* = 7.0），而向酰基酶的分解在高pH值下是速率决定步骤。校正温度和共溶剂的影响后，中间体形成的速率与通过停流技术在25℃水溶液中测得的速率高度一致。
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