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

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:
OSTI Identifier:
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
Research Org:
Institute of Organic Chemistry and Biochemistry, Prague, Czechoslovakia
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