Designing of Substrates and Inhibitors of Bovine α-Chymotrypsin with Synthetic Phenylalanine Analogues in Position P1

Abstract

The primary specificity residue of a substrate or an inhibitor, called the P(1) residue, is responsible for the proper recognition by the cognate enzyme. This residue enters the S(1) 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 alpha-chymotrypsin substrate activity, aromatic non-proteinogenic amino acid residues in position P(1) with the sequence Ac-Phe-Ala-Thr-X-Anb(5,2)-NH(2) were introduced: L-pyridyl alanine (Pal), 4-nitrophenylalanine - Phe(p-NO(2)), 4-aminophenylalanine - Phe(p-NH(2)), 4-carboxyphenylalanine Phe(p-COOH), 4-guanidine phenylalanine - Phe(p-guanidine), 4-methyloxycarbonyl-phenylalanine - 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 (k(cat), K(M) and k(cat)/K(M)) of the peptides synthesized with bovine alpha-chymotrypsin were determined. The highest value of the specificity constant k(cat)/K(M), reaching 6.0 x 10(5) [M(-1)xs(-1)], was obtained for Ac-Phe-Ala-Thr-Phe(p-NO(2))-Anb(5,2)-NH(2). The replacement of the acetyl group with benzyloxycarbonyl moiety yielded a substrate with the value of k(cat) more than three times higher. Peptide aldehydes were synthesized with selected residues (Phe, Pal, Tyr, Phe(p-NO(2)) in position P(1) and potent chymotrypsin inhibitors were obtained. The dissociation constant (K(i)) with the experimental enzyme determined for the most active peptide, Tos-Phe-Ala-Thr-Phe(p-NO(2))-CHO, amounted to 1.12 x 10(-8) M.

Proteolysis inside the Membrane Is a Rate-Governed Reaction Not Driven by Substrate Affinity

“…It’s important to note that it’s not the absolute K M value itself that leads to this conclusion: while some proteases like chymotrypsin also display high K M values (Wysocka et al, 2008), these digestive proteases encounter food protein at extraordinarily high and thus matched concentrations. Instead, although there is little precedent for interpreting binding affinities within 2-dimensions, a K M of ~0.14 mole percent is extraordinarily high, because the inner membrane of E. coli contains only 1.25–1.67 mole percent (~50% by weight) of total protein (Schnaitman, 1970a, b).…”

Non-Canonical Amino Acids in Analyses of Protease Structure and Function

“…In addition, a quantitative structure–activity relationship (QSAR) was derived for the substrate fragments, i.e., essentially, the individual residues and overall log (k cat /K M ) values of the substrates resulted from additive contributions of the fragments [ 133 ]. Based on these data, CTRA aldehyde inhibitors were synthesized with modified Phe residues in the P1 position, of which Tos-Phe-Ala-Thr-Phe(p-NO 2 )-CHO had a K i of 11 nM [ 134 ]. A previous investigation on the chymotrypsin-like Streptomyces griseus protease B (SGPB, S01.262) with the uncommonly favored P1-Glu analyzed the interaction with the 55-residue-long third domain of the turkey ovomucoid inhibitor (OMTKY3, I01.003) using a homologous series of the aliphatic P1-side chains Gly, Ala, Abu, Ape (Nva), α-Ahx (Nle) and Ahp ( Figure 1 ) [ 135 ].…”