Abstract
Fluorescence studies on the interaction, with porcine pepsin, of oligopeptides bearing a mansyl (Mns, 6-(N-methylanilino)-2-naphthalenesulfonyl) or dansyl (Dns, 5-dimethylaminonaphthalene-1-sulfonyl) group at the NH2 or COOH terminus have provided further evidence showing that the probe group is drawn into the extended active site largely as a consequence of the specific binding of the peptide portion of the substrate. The active site does not appear to have appreciable intrinsic affinity for the mansyl or dansyl group, and the principal contribution to the specific peptide-protein interaction is provided by the sensitive L-phenylalanyl-L-phenylalanyl (Phe-Phe) unit of the substrates tested. The pepsin inhibitor pepstatin can displace substrates such as Mns-(Gly)n-Phe-Phe-OR or Gly-Gly-Phe-Phe-NHNH-Mns from the active site of porcine pepsin; in these circumstances the mansyl group is bound weakly at a separate, nonspecific locus, distinct from the active site, which can accept the mansyl group of Mns-Gly-Gly-OR or mansylamide. In the interaction with substrates such as Mns-(Gly)n-Phe-Phe-OR or Dns-(Gly)n-Phe-Phe-OR, the above conclusions for porcine pepsin also apply to Rhizopus-pepsin. With substrates such as Gly-Gly-Phe-Phe-NHNH-Mns, however, the active site of Rhizopus-pepsin shows less affinity for the fluorescent probe group than does that of porcine pepsin, suggesting structural differences between the two acid proteinases in the region of their extended active sites which bind the COOH-terminal portion of small oligopeptide substrates.
Highlights
Dns-(Gly),Phe-Phe-OR, the above conclusions for porcine pepsin apply to Rhizopus-pepsin. With substrates such as Gly-Gly-Phe-Phe-NHNH-Mns, the active site of Rhizopus-pepsin shows less affinity for the fluorescent probe group than does that of porcine pepsin, suggesting structural differences between the two acid proteinases in the region of their extended active sites which bind the COOH-terminal portion of small oligopeptide substrates
In t.he experiments in which mansyl or dansyl peptide substrates were in the presence of equimolar or excess enzyme, the intensity and position of the emission maximum was determined within 1 min of mixing the reactants
Because of the negligible fluorescence of the unbound mansyl compound at the wavelength of the emission maximum of the protein-ligand complex, the fluorescence intensity at that wavelength could be used as a measure of the concentration of the complex
Summary
Fluorescence studies on the interaction, with porcine pepsin, of oligopeptides bearing a mansyl Methylanilino)-Z-naphthalenesulfonyl) or dansyl (Dns, 5-dimethylaminonaphthalene-1-sulfonyl) group at the NH, or COOH terminus have provided further evidence showing that the probe group is drawn into the extended active site largely as a consequence of the specific binding of the peptide portion of the substrate. (Gly),-Phe-Phe-OR or Dns-(Gly),-Phe-Phe-OR, the above conclusions for porcine pepsin apply to Rhizopus-pepsin With substrates such as Gly-Gly-Phe-Phe-NHNH-Mns, the active site of Rhizopus-pepsin shows less affinity for the fluorescent probe group than does that of porcine pepsin, suggesting structural differences between the two acid proteinases in the region of their extended active sites which bind the COOH-terminal portion of small oligopeptide substrates. Data are offered in favor of the view that Mns-Gly-Gly-01’4P is bound largely at the mansylamide-binding site, and not at the active site of pepsin These dat’a are consistent with the suggestion [2] that the marlsylamide~birldin~ site is accessible to the mansyl group of substrates of the type A-l’he-l’he-01’41’. That this interaction is observed when the active site is blocked, as in pepsinogen, through inhibition by pepstatin or by a specific chemical modification of an active site carbosyl group with tosyl-L-phenylalanyldiazomethane [6] to inactivate the enzyme
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