Hunkapiller, Michael W. and Forgac, Michael D. and Richards, John H. (1976) Mechanism of action of serine proteases: tetrahedral intermediate and concerted proton transfer. Biochemistry, 15 (25). pp. 5581-5588. ISSN 0006-2960. doi:10.1021/bi00670a024. https://resolver.caltech.edu/CaltechAUTHORS:20150505-094437203
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Abstract
Stopped-flow spectrophotometry and proton inventory experiments have been used to define the reaction pathway for hydrolysis of a specific peptide substrate, Ac-L-Ala-L-Pro-L-Ala p-nitroanilide, by the serine proteases elastase and α-lytic protease. The stopped-flow studies reveal the existence and buildup of a tetrahedral adduct between the active site serine hydroxyl group and the sensitive carbonyl group of the substrate. The decomposition of this tetrahedral intermediate to the acyl enzyme and p-nitroaniline is the rate-limiting step for the hydrolytic reaction. The proton inventory data suggest the simultaneous transfer of two protons (presumably from the catalytic carboxyl of Asp-102 to Nπ of the catalytic imidazole of His-57 and from Nτ of the imidazole to the anilide NH) in the transition state leading to breakdown of the tetrahedral complex. That these proton transfers occur in a concerted, rather than stepwise, process attests to the ability of enzymes to lower the enthalpy of activation most effectively when the precise alignment of a highly specific substrate and catalytic groups minimizes the entropy of activation.
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Additional Information: | © 1976 American Chemical Society. This work was supported by a grant from the National Institutes of Health (GM 10218). National Institutes of Health Postdoctoral Fellow. We gratefully acknowledge Professor R. L. Showen for numerous discussions on the proton inventory experiments and for providing us with preprints of his work. We are also indebted to Professor H. B. Gray and S. E. Wherland for their assistance in the stopped-flow kinetic studies and use of their Durrum spectrophotometer. | |||||||||
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Issue or Number: | 25 | |||||||||
DOI: | 10.1021/bi00670a024 | |||||||||
Record Number: | CaltechAUTHORS:20150505-094437203 | |||||||||
Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:20150505-094437203 | |||||||||
Official Citation: | Mechanism of action of serine proteases: tetrahedral intermediate and concerted proton transfer Michael W. Hunkapiller, Michael D. Forgac, and John H. Richards Biochemistry 1976 15 (25), 5581-5588 DOI: 10.1021/bi00670a024 | |||||||||
Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | |||||||||
ID Code: | 57222 | |||||||||
Collection: | CaltechAUTHORS | |||||||||
Deposited By: | Tony Diaz | |||||||||
Deposited On: | 05 May 2015 19:48 | |||||||||
Last Modified: | 10 Nov 2021 21:09 |
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