Enzymatic semisynthesis of aprotinin homologues mutated in P′ positions

Abstract

The replacement of amino acids in the P′₁ and P′₂ position of aprotinin, the bovine pancreatic trypsin inhibitor, is described. Using the “modified” inhibitor as starting material, with the hydrolyzed reactive-site peptide bond Lys¹⁵-Ala¹⁶, the residues P′₁ (Ala¹⁶) and P′₂ (Arg¹⁷) were split off by the action of aminopeptidase K. Incorporation of suitable dipeptides containing a basic residue (Lys or Arg) in the C-terminal position was carried out in a “one pot” reaction involving trypsin-catalyzed coupling. In this way, the native fragment Ala¹⁶-Arg¹⁷ was reintroduced and also replaced by Gly-Arg, Ala-Lys, and Leu-Arg yielding intact inhibitor molecules. The mechanism for incorporation of dipeptides was investigated by treating the aprotinin derivative with the Arg¹⁷-Ile¹⁸ peptide bond hydrolyzed with trypsin under proteosynthetic conditions. We established that only inhibitor molecules cleaved between Lys¹⁵ and Xaa¹⁶ are intermediates leading to the desired products. The inhibitory properties of the new aprotinin homologues were tested, and the significance of the P′₁ residue for the inhibition of trypsin, kallikrein, and chymotrypsin was deduced.