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Carbon nuclear magnetic resonance studies of the histidine residue in α-lytic protease. Implications for the catalytic mechanism of serine proteases

Hunkapiller, Michael W. and Smallcombe, Stephen H. and Whitaker, Donald R. and Richards, John H. (1973) Carbon nuclear magnetic resonance studies of the histidine residue in α-lytic protease. Implications for the catalytic mechanism of serine proteases. Biochemistry, 12 (23). pp. 4732-4743. ISSN 0006-2960. doi:10.1021/bi00747a028. https://resolver.caltech.edu/CaltechAUTHORS:20150505-104057052

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Abstract

Selective ^(13)C enrichment of C-2 of the single histidine residue of the serine protease α-lytic protease has allowed direct study of the Asp-His-Ser catalytic triad. Both the chemical shift of C-2 and the coupling between C-2 and its directly bonded hydrogen have been observed as a function of pH. We interpret the results to indicate that only below pH 4 does the imidazole ring of the histidine residue become protonated and only above pH 6.7 does the aspartic acid residue lose a proton to generate a carboxylate anion. Thus, over the pH range 4-6.7, the catalytic triad consists of a neutral aspartic acid and a neutral histidine residue-not the ionized forms hitherto assumed. These new assignments for the ionization characteristics of the aspartic acid and histidine residues of the catalytic triad lead to a proposed catalytic mechanism that avoids any requirement for unfavorable charge separation. In this view, the histidine residue plays two roles: (i) it provides insulation between water and the buried carboxylate anion of the aspartate, thus ensuring the carboxylate group a hydrophobic environment, and (ii) it provides a relay for net transfer of protons from the serine hydroxyl to the carboxylate anion. The aspartate anion acts as the ultimate base which holds a proton during catalysis. An anionic, rather than a neutral, base has advantages; it both avoids the necessity of charge separation and, by giving the catalytic locus an overall negative charge, assists preferential expulsion of product relative to substrate from the active site of the enzyme. Relaxation measurements (T_I, T_2, and nuclear Overhauser enhancement) indicate that, over the pH range of enzymic activity, the histidine residue is held rigidly within the protein.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/bi00747a028DOIArticle
http://pubs.acs.org/doi/abs/10.1021/bi00747a028PublisherArticle
Additional Information:© 1973 American Chemical Society. Received June 13, 1973. Contribution No. 4699 from the Church Laboratories of Chemical Biology, California Institute of Technology, Pasadena, California 91109. Received June 13, 1973. We wish to acknowledge generous financial support of this research by grants from the U. S. Public Health Service, NIGMS-16424 and NIGMS-10218, and the Medical Research Council of Canada, MA-4409.
Funders:
Funding AgencyGrant Number
U. S. Public Health Service (USPHS)NIGMS-16424
U. S. Public Health Service (USPHS)NIGMS-10218
Medical Research Council of CanadaMA-4409
Other Numbering System:
Other Numbering System NameOther Numbering System ID
Caltech Division of Chemistry and Chemical Enginering4699
Issue or Number:23
DOI:10.1021/bi00747a028
Record Number:CaltechAUTHORS:20150505-104057052
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20150505-104057052
Official Citation:Hunkapiller, M. W., Smallcombe, S. H., Whitaker, D. R., & Richards, J. H. (1973). Carbon nuclear magnetic resonance studies of the histidine residue in α-lytic protease. Implications for the catalytic mechanism of serine proteases. Biochemistry, 12(23), 4732-4743. doi: 10.1021/bi00747a028
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:57228
Collection:CaltechAUTHORS
Deposited By: Joanne McCole
Deposited On:05 May 2015 19:52
Last Modified:10 Nov 2021 21:09

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