Isolation and characterization of a protease inhibitor from Acacia karroo with a common combining loop and overlapping binding sites for chymotrypsin and trypsin

• A Kunitz-P inhibitor was isolated and characterized from seeds of Acacia karroo.
• AkCI/1 inhibits strongly (and almost equally) trypsin and chymotrypsin.
• We modeled the interactions of trypsin and chymotrypsin with the inhibitor.
• Our studies suggest that AkCI/1 uses overlapping binding sites for the two proteases.
• The canonical loop has to undergo a conformational change upon chymotrypsin binding.

By using affinity and reversed-phase HPLC (RP-HPLC) chromatographies two chymotrypsin–trypsin inhibitors were isolated from seeds of Acacia karroo, a legume of the subfamily Mimosoideae. The primary structure of one of these inhibitors, named AkCI/1, was determined. The inhibitor consists of two polypeptide chains, 139 and 44 residues respectively, which are linked by a single disulfide bridge. The amino acid sequence of AkCI/1 is homologous to and showed more than 60% sequence similarity with other protease inhibitors isolated earlier from the group of Mimosoideae. AkCI/1 inhibits both chymotrypsin (EC 3.4.21.1) and trypsin (EC 3.4.21.4) in a 1:1 M ratio with Ki values of 2.8 × 10−12 M and 1.87 × 10−12 M, respectively. The P1–P1′ residues for trypsin were identified as Arg68-Ile69 by selective hydrolysis of the inhibitor at this site, with bovine trypsin and human trypsin IV. The cleavage did not affect the inhibition of trypsin, but fully abolished the chymotrypsin inhibitory activity of AkCI/1. This finding together with our studies on competition of the two enzymes for the same combining loop suggests that the same loop has to contain the binding sites for both proteases. The most likely P1 residue of AkCI/1 for chymotrypsin is Tyr67.