title: Cleavage of the X-Pro peptide bond by pepsin is specific for the trans isomer.
abstract: Fluorine nuclear magnetic resonance studies of the cleavage of peptides containing a 4-fluorophenylalanine (FPhe)-Pro bond have been performed in order to determine the conformational specificity of FPhe-Pro bond cleavage by pepsin. The peptides selected were substrates of HIV protease or of avian sarcoma virus protease, both of which have been reported to be cleaved specifically at X-Pro by pepsin as well as by the corresponding viral protease enzyme. By working at 0 degrees C, it was possible to separate kinetically cleavage and cis/trans isomerization. For the case of the protease substrate, Ser-Gln-Asn-FPhe-Pro-Ile-Val-Gln, cleavage was shown to be specific for the trans conformation. A value for the rate constant for hydrolysis of the trans peptide divided by the Michaelis constant, ktH/KMtrans = 0.3 min-1 mM-1 was obtained with this substrate, and the Michaelis constant appears to be considerably higher than the substrate concentration, 3.7 mM, used in the study. On a slower time scale, additional cleavages can readily be detected. For the avian leukemia virus protease substrate, Thr-Phe-Gln-Ala-FPhe-Pro-Leu-Arg-Glu-Ala, the cleavage was both slower and less specific. In addition to the primary cleavage at the FPhe-Pro site, cleavage also occurs at the Ala-FPhe bond on a somewhat slower time scale. In addition to the conformational specificity of the cleavage reaction, these results indicate that pepsin is a better model for HIV protease than for avian leukemia virus protease.
authors.affiliation: Laboratory of Structural Biology, NIEHS, Box 12233, Research Triangle Park, North Carolina 27709, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Vance
authors.forename: D A
authors.initials: DA
authors.lastname: LeBlanc
authors.forename: R E
authors.initials: RE
authors.lastname: London
mesh_terms: D006868:Hydrolysis; D007536:Isomerism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009842:Oligopeptides / Q000378:metabolism*; D010434:Pepsin A / Q000378:metabolism*; D011392:Proline / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity; D010135:p-Fluorophenylalanine / Q000378:metabolism
chemical_list: D009842:Oligopeptides; D010135:p-Fluorophenylalanine; D011392:Proline; D010434:Pepsin A