N-Methyl-2-anilinonaphthalene-6-sulfonyl Peptides as Fluorescent Probes for Pepsin-Substrate Interaction

• Published in: The Journal of biological chemistry 1973-09, Vol.248 (18), p.6292-6299
• Main Authors: Sachdev, Goverdhan P., Brownstein, Allen D., Fruton, Joseph S.
• Format: Article
• Language: English
• Subjects: Aniline Compounds - chemical synthesis; Binding Sites; Chromatography; Chromatography, Paper; Fluorescence; Kinetics; Naphthalenes - chemical synthesis; Pepsin A; Peptides; Protein Binding; Silicon Dioxide; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Sulfonic Acids; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)43447-9

Advantage has been taken of the high sensitivity of the N-methyl-2-anilinonaphthalene-6-sulfonyl (mansyl) group as a fluorescent probe for hydrophobic regions of proteins to study the interaction of pepsin with mansylamide and with several peptides bearing an amino-terminal mansyl group. The data indicate that pepsin has a binding site for mansylamide that is distinct from the substrate-binding region of the enzyme. When a mansyl group is attached to a peptide having an l-phenylalanyl-l-phenylalanyl bond, previously identified as a preferred linkage for attack by pepsin, the fluorescent probe appears to be drawn into a hydrophobic region not evident in the interaction with mansylamide. Upon the addition of pepstatin, a powerful competitive inhibitor of pepsin, the enhancement of the fluorescence of mansylamide is unaffected, but that of the mansyl peptides is reduced to the value for mansylamide. Further evidence for the discrete nature of the binding sites in pepsin for mansylamide and for the mansyl group of peptide substrates is provided by the fact that, upon autocatalytic activation of pepsinogen, the enhancement of the fluorescence of mansylamide is decreased, whereas that of the mansyl peptides is increased; both effects are abolished by pepstatin. Studies on the fluorescence of mansylamide and of mansyl peptides in the presence of pepsin that had been stoichiometrically inactivated by treatment with tosyl-l-phenyl-alanyldiazomethane have shown that this active site-directed inhibitor not only blocks the access of a mansyl peptide substrate to the active site, but also alters the mansylamide-binding site so as to lower its polarity. Further evidence for the conformational flexibility of pepsin suggested by this finding is provided by data on the partially acetylated enzyme, which is more active toward oligopeptide substrates than is pepsin itself. With acetyl-pepsin, the enhancement of the fluorescence of mansyl peptides (but not of mansylamide) is much greater than with untreated pepsin. The addition of pepstatin, however, markedly alters the polarity of the mansylamide-binding site of acetyl-pepsin. Data are presented for the kinetics of the cleavage, by pepsin, of several mansyl peptide substrates. These results, together with those from the fluorescence studies, give further evidence of the importance of secondary interactions of pepsin substrates with the extended active site of the enzyme in influencing catalytic efficiency without marked chan