Substitutions at the P-1 ' 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 bovin e pancreatic trypsin inhibitor (BPTI); the mutants contain Gly, Ala, Ser, V al, Leu, Arg, and Trp at the P-1' position of the reactive site. The effect s of substitutions at the P-1' position on the association constants are ve ry large, comprising seven orders of magnitude for trypsin and plasmin, and over five orders for chymotrypsin. All mutants showed a decrease of the as sociation constant to the three proteinases in the same order: Ala > Gly > Ser > Arg > Val > Leu > Trp. Calorimetric and circular dichroism methods sh owed that none of the P1' substitutions, except the P1'-Val mutant, lead to destabilisation of the binding loop conformation. The X-ray structure of t he 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 th ereby is forced to rotate approximately 90 degrees. lle18 (P-3') in its new orientation, in turn interacts with the Tyr39 side-chain of trypsin. Intro duction of a large side-chain at the P1' position apparently leads to a cas cade of small alterations of the trypsin-BPTI interface that seem to destab ilise the complex by it adopting a less optimized packing and by tilting th e BPTI molecule up to 15 degrees compared to the native trypsin-BPTI comple x. (C) 2000 Academic Press.