CaltechAUTHORS
  A Caltech Library Service

Catalytic Mechanism of Serine Proteases: Reexamination of the pH Dependence of the Histidyl 1J{13C2-H} Coupling Constant in the Catalytic Triad of alpha-lytic Protease

Bachovchin, William W. and Kaiser, Robert and Richards, John H. and Roberts, John D. (1981) Catalytic Mechanism of Serine Proteases: Reexamination of the pH Dependence of the Histidyl 1J{13C2-H} Coupling Constant in the Catalytic Triad of alpha-lytic Protease. Proceedings of the National Academy of Sciences of the United States of America, 78 (12). pp. 7323-7326. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:BACpnas81

[img]
Preview
PDF
See Usage Policy.

562Kb

Use this Persistent URL to link to this item: http://resolver.caltech.edu/CaltechAUTHORS:BACpnas81

Abstract

L-Histidine, 90% 13C enriched at the C2 position, was incorporated into the catalytic triad of alpha-lytic protease (EC 3.4.21.12) with the aid of a histidine-requiring mutant of Lysobacter enzymogenes (ATC 29487), and the pH dependence of the coupling constant between this carbon atom and its directly bonded proton was reinvestigated. The high degree of specific 13C isotopic enrichment attainable with the auxotroph permits direct observation and measurement of this coupling constant in proton-coupled 13C NMR spectra at 67.89 MHz and at 15.1 MHz. In contrast to the earlier study, the present results indicate that this coupling constant does respond to a microscopic ionization with pKa near 7.0; moreover, the magnitude of the values of 1JC-H observed are in accord with those expected for titration of the histidyl residue. We conclude that the original measurement must be in error and that this coupling constant now also supports a histidyl residue that titrates more or less normally as a component of the catalytic triad of serine proteases.


Item Type:Article
Additional Information:Copyright © 1981 by the National Academy of Sciences. Contributed by John D. Roberts, August 10, 1981. This work was supported by grants from the National Institutes of Health (GM-27927 and GM164221) and from Research Corporation. The high-field NMR experiments were performed at the NMR Facility for Biomolecular Research located at the F. Bitter National Magnet Laboratory (Massachusetts Institute of Technology). The NMR Facility is supported by Grant RR00995 from the Division of Research Resources of the National Institutes of Health and by National Science Foundation Contract C-670. Presented in part at the Ninth International Conference on Magnetic Resonance in Biological Systems, Bendor, France, September 1-6, 1980.
Subject Keywords:13C NMR, enzyme mechanisms, biosynthetic isotopic enrichment, histidine auxotroph, charge-relay system
Record Number:CaltechAUTHORS:BACpnas81
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:BACpnas81
Alternative URL:http://www.pnas.org/cgi/content/abstract/78/12/7323
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:8936
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:03 Oct 2007
Last Modified:26 Dec 2012 09:43

Repository Staff Only: item control page