PMID: 36609Apr 1, 1979

Detection and accumulation of tetrahedral intermediates in elastase catalysis

Proceedings of the National Academy of Sciences of the United States of America
A L Fink, P Meehan


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.


Jan 1, 1972·Cold Spring Harbor Symposia on Quantitative Biology·R HendersonD M Blow
Apr 18, 1973·Journal of the American Chemical Society·E C LucasK J Bush
May 17, 1972·Journal of the American Chemical Society·M H O'Leary, M D Kluetz

Related Concepts

Protein Conformation
Elastase I
Aqueous solution
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Canis familiaris
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