Substitutions at the P1′ position in BPTI strongly affect the association energy with serine proteinases

The role of the S-1' subsite in trypsin, chymotrypsin and plasmin has been examined by measuring the association with seven different mutants of bovine pancreatic trypsin inhibitor (BPTI); the mutants contain Gly, Ala, Ser, Val, Leu, Arg, and Trp at the P-1' position of the reactive site. The effects of substitutions at the P-1' position on the association constants are very large, comprising seven orders of magnitude for trypsin and plasmin, and over five orders for chymotrypsin. All mutants showed a decrease of the association constant to the three proteinases in the same order: Ala > Gly > Ser > Arg > Val > Leu > Trp. Calorimetric and circular dichroism methods showed that none of the P1' substitutions, except the P1'-Val mutant, lead to destabilisation of the binding loop conformation. The X-ray structure of the complex formed between bovine beta-trypsin and P-1'-Leu BPTI showed that the P-1'-Leu sterically conflicts with the sidechain of P-3'-Ile, which thereby is forced to rotate approximately 90 degrees. lle18 (P-3') in its new orientation, in turn interacts with the Tyr39 side-chain of trypsin. Introduction of a large side-chain at the P1' position apparently leads to a cascade of small alterations of the trypsin-BPTI interface that seem to destabilise the complex by it adopting a less optimized packing and by tilting the BPTI molecule up to 15 degrees compared to the native trypsin-BPTI complex. (C) 2000 Academic Press.