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Title: Evidence for segmental mobility in the active site of pepsin

Abstract

The low hydrolytic activity (k/sub cat/ < 0.001 s/sup -1/) of chicken pepsin (CP) towards tri- and tetrapeptides is enhanced at least 100 times by modification of its single sulfhydryl group of Cys-115, with little effect on K/sub m/-values. Modification thus simulates the effect of secondary substrate binding on pepsin catalysis. The rate of Cys-115 modification is substantially decreased in the presence of some competitive inhibitors, suggesting its active site location. Experiments with CP alkylated at Cys-115 with Acrylodan as a fluorescent probe or with N-iodoacetyl-(4-fluoro)-aniline as a /sup 19/F-nmr probe suggest conformation change around Cys-115 to occur on substrate or substrate analog binding. The difference /sup 1/H-nmr spectra (500 MHz) of unmodified free and inhibitor-complexed CP reveal chemical shifts almost exclusively in the aromatic region. The effects of Cu/sup + +/ on /sup 19/F- and /sup 1/H-nmr spectra have been studied. Examination of a computer graphics model of CP based on E. parasitica pepsin-inhibitor complex X-ray coordinates suggests that Cys-115 is located near the S/sub 3//S/sub 5/ binding site. The results are interpreted in favor of segmental mobility of this region important for pepsin substrate binding and catalysis.

Authors:
; ; ; ;
Publication Date:
Research Org.:
Institute of Organic Chemistry and Biochemistry, Prague, Czechoslovakia
OSTI Identifier:
5327045
Alternate Identifier(s):
OSTI ID: 5327045
Report Number(s):
CONF-8606151-
Journal ID: CODEN: FEPRA
Resource Type:
Conference
Resource Relation:
Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States); Journal Volume: 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 62 RADIOLOGY AND NUCLEAR MEDICINE; PEPSIN; ENZYME ACTIVITY; RECEPTORS; BIOCHEMICAL REACTION KINETICS; ANILINE; CATIONS; CHEMICAL SHIFT; CHICKENS; COPPER COMPOUNDS; FLUORINE 19; NMR SPECTRA; NUCLEAR MAGNETIC RESONANCE; ACID PROTEINASES; AMINES; ANIMALS; AROMATICS; BIRDS; CHARGED PARTICLES; ENZYMES; FLUORINE ISOTOPES; FOWL; HYDROLASES; IONS; ISOTOPES; KINETICS; LIGHT NUCLEI; MAGNETIC RESONANCE; MEMBRANE PROTEINS; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; PEPTIDE HYDROLASES; PROTEINS; REACTION KINETICS; RESONANCE; SPECTRA; STABLE ISOTOPES; TRANSITION ELEMENT COMPOUNDS; VERTEBRATES 550201* -- Biochemistry-- Tracer Techniques; 550600 -- Medicine

Citation Formats

Pohl, J., Strop, P., Senn, H., Foundling, S., and Kostka, V.. Evidence for segmental mobility in the active site of pepsin. United States: N. p., 1986. Web.
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Pohl, J., Strop, P., Senn, H., Foundling, S., & Kostka, V.. Evidence for segmental mobility in the active site of pepsin. United States.
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Pohl, J., Strop, P., Senn, H., Foundling, S., and Kostka, V.. Thu . "Evidence for segmental mobility in the active site of pepsin". United States. doi:.
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@article{osti_5327045,
title = {Evidence for segmental mobility in the active site of pepsin},
author = {Pohl, J. and Strop, P. and Senn, H. and Foundling, S. and Kostka, V.},
abstractNote = {The low hydrolytic activity (k/sub cat/ < 0.001 s/sup -1/) of chicken pepsin (CP) towards tri- and tetrapeptides is enhanced at least 100 times by modification of its single sulfhydryl group of Cys-115, with little effect on K/sub m/-values. Modification thus simulates the effect of secondary substrate binding on pepsin catalysis. The rate of Cys-115 modification is substantially decreased in the presence of some competitive inhibitors, suggesting its active site location. Experiments with CP alkylated at Cys-115 with Acrylodan as a fluorescent probe or with N-iodoacetyl-(4-fluoro)-aniline as a /sup 19/F-nmr probe suggest conformation change around Cys-115 to occur on substrate or substrate analog binding. The difference /sup 1/H-nmr spectra (500 MHz) of unmodified free and inhibitor-complexed CP reveal chemical shifts almost exclusively in the aromatic region. The effects of Cu/sup + +/ on /sup 19/F- and /sup 1/H-nmr spectra have been studied. Examination of a computer graphics model of CP based on E. parasitica pepsin-inhibitor complex X-ray coordinates suggests that Cys-115 is located near the S/sub 3//S/sub 5/ binding site. The results are interpreted in favor of segmental mobility of this region important for pepsin substrate binding and catalysis.},
doi = {},
journal = {Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)},
number = ,
volume = 45:6,
place = {United States},
year = {Thu May 01 00:00:00 EDT 1986},
month = {Thu May 01 00:00:00 EDT 1986}
}
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