title: Design, chemical synthesis and kinetic studies of trypsin chromogenic substrates based on the proteinase binding loop of Cucurbita maxima trypsin inhibitor (CMTI-III).
abstract: A series of trypsin chromogenic substrates with formula: Y-Ala-X-Abu-Pro-Lys-pNA, where X = Gly, Ala, Abu, Val, Leu, Phe, Ser, Glu and Y = Ac, H; pNA = p-nitroanilide was synthesized. The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design the trypsin substrates. To evaluate the influence of position P(4) on the substrate-enzyme interaction, kinetic parameters of newly synthesized substrates with bovine beta-trypsin were determined. The increasing hydrophobicity of the amino acid residue (Gly, Ala, Abu, Val) introduced in position P(4) significantly enhanced the substrate specificity (k(cat)/K(m)) which was over 8 times higher for the last residue than that for the first one. The introduction of residues with more hydrophilic side chain (Glu, Ser) in this position reduced the value of this parameter. These results correspond well with those obtained using molecular dynamics of bovine beta-trypsin with monosubstituted CMTI-I analogues, indicating that in both trypsin substrate and inhibitor position 4 plays an important role in the interaction with the enzyme.
authors.affiliation: Faculty of Chemistry, University of Gdańsk, Gdańsk, PL-80-502, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Lesner
authors.forename: K
authors.initials: K
authors.lastname: Brzozowski
authors.forename: G
authors.initials: G
authors.lastname: Kupryszewski
authors.forename: K
authors.initials: K
authors.lastname: Rolka
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002863:Chromogenic Compounds; D019662:Cucurbitaceae / Q000737:chemistry; D015195:Drug Design; D004355:Drug Stability; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism
chemical_list: C081870:CMTI protein, Cucurbita maxima; D002863:Chromogenic Compounds; D010940:Plant Proteins; D014361:Trypsin Inhibitors; D014357:Trypsin