Abstract
The primary specificity residue of a substrate or an inhibitor, called the P1 residue, is responsible for the proper recognition by the cognate enzyme. This residue enters the S1 pocket of the enzyme and establishes contacts (up to 50%) inside the proteinase substrate cavity, strongly affecting its specificity. To analyze the influence on bovine α-chymotrypsin substrate activity, aromatic non-proteinogenic amino acid residues in position P1 with the sequence Ac-Phe-Ala-Thr-XAnb 5,2-NH2 were introduced: L-pyridyl alanine (Pal), 4-nitrophenylalanine – Phe(p-NO2), 4-aminophenylalanine – Phe(p- NH2), 4-carboxyphenylalanine Phe(p-COOH), 4-guanidine phenylalanine – Phe(p-guanidine), 4-methyloxycarbonylphenylalanine – Phe(p-COOMe), 4-cyanophenylalanine – Phe(p-CN), Phe, Tyr. The effect of the additional substituent at the phenyl ring of the Phe residue was investigated. All peptides contained an amide of 5-amino-2-nitrobenzoic acid, which served as a chromophore. Kinetic parameters (kcat, KM and kcat/KM) of the peptides synthesized with bovine α- chymotrypsin were determined. The highest value of the specificity constant kcat/KM, reaching 6.0x105 [M-1xs-1], was obtained for Ac-Phe-Ala-Thr-Phe(p-NO2)-Anb5,2-NH2. The replacement of the acetyl group with benzyloxycarbonyl moiety yielded a substrate with the value of kcat more than three times higher. Peptide aldehydes were synthesized with selected residues (Phe, Pal, Tyr, Phe(p-NO2) in position P1 and potent chymotrypsin inhibitors were obtained. The dissociation constant (Ki) with the experimental enzyme determined for the most active peptide, Tos-Phe-Ala-Thr-Phe(p-NO2)-CHO, amounted to 1.12x10-8 M.
Keywords: chromogenic substrate, chymotrypsin, peptide aldehyde, peptidomimetic
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