Binding of native and [homoserine lactone‐52]‐52,53‐seco‐bovine basic pancreatic trypsin inhibitor (kunitz inhibitor) to porcine pancreatic β‐kallikrein‐B and bovine α‐chymtorpsin: Thermodynaic study

Abstract

Values of the association equilibrium constant (Ka) for the binding of the native and of the cyanogen bromide-cleaved bovine basic pancreatic trypsin inhibitor (native BPTI and [Hse lactone-52]-52,53-seco-BPTI, respectively) to neuraminidase-treated porcine pancreatic beta-kallikrein-B (kallikrein) and bovine alpha-chymotrypsin (chymotrypsin) have been determined between pH 4.0 and 9.0, at 20.0 degrees C. Over the whole pH range explored, native BPTI and [Hse lactone-52]-52,53-seco-BPTI show the same affinity for kallikrein. On the other hand, the affinity of [Hse lactone-52]-52,53-seco-BPTI for chymotrypsin is higher, around neutrality, than that found for native BPTI by about one order of magnitude, converging in the acidic pH limb. The simplest mechanism accounting for the observed data implies that, on lowering the pH from 9.0 to 4.0, (i) the decrease in affinity for the binding of native BPTI to kallikrein and chymotrypsin, as well as for the association of [Hse lactone-52]-52,53-seco-BPTI to kallikrein, reflects the acidic pK shift, upon inhibitor association, of a single ionizing group; and (ii) the decrease of Ka values for [Hse lactone-52]-52,53-seco-BPTI binding to chymotrypsin appears to be modulated by the acidic pK shift, upon inhibitor association, of two non-equivalent proton-binding residues. On the basis of the stereochemistry of the serine proteinase/inhibitor contact region(s), these data indicate that long-range structural changes in [Hse lactone-52]-52,53-seco-BPTI are energetically linked to the chymotrypsin:inhibitor complex formation. This observation represents an important aspect for the mechanism of molecular recognition and regulation in BPTI.

Crystal structure of the bovine α-chymotrypsin:kunitz inhibitor complex. An example of multiple protein:protein recognition sites

“…In this respect, it can be noted that, despite the close location in the complex structure, the Lys315 side chain leaves enough room on the ␣-CHT surface for residue Met192, whose oxidation to sulphoxide does not affect BPTI binding to the serine proteinase (Cutruzzolà et al, 1993). Moreover, the location of the guanidino group of residue Arg317(P 2 Ј), close to the side chain of the ␣-CHT residue His40, is in keeping with its proposed role as a stabilizing residue in the formation of the ␣-CHT complex with [homoserine lactone-52]-52,53-seco-BPTI (Oddone et al, 1994).…”

“…As a first step, in the structural analysis, the self-rotation function was calculated in the 15.0-3.5 Å resolution range, locating a prominent peak at = 26°, = 90°, = 180°, with a correlation coeficient of 67.4 (Navaza, 1994). Next, the cross rotational search was run employing as search molecule a locally built model of the ␣-CHT:BPTI complex (Cutruzzolà et al, 1993;Oddone et al, 1994), based on the superposition of ␣-CHT [monomer A from the dimeric ␣-CHT structure; PDB code 2CHA (Tsukada and Blow, 1985)] onto the ␤-TRP:BPTI complex [PDB code 2PTC (Rühlmann et al, 1973)]. The rotational search was run in the 15.0-3.5 Å resolution range, providing two solutions (2.5 r.m.s.…”