Carbon-13 nuclear magnetic resonance studies of the selectively isotope-Labeled reactive site peptide bond of the basic pancreatic trypsin inhibitor in the complexes with trypsin, trypsinogen, and anhydrotrypsin

Abstract

A previously characterized modification of the basic pancreatic trypsin inhibitor (BPTI), with the carbonyl carbon atom of Lys-15 selectively enriched in 13C, the peptide bond Arg-39-Ala-40 cleaved and Arg-39 removed, was used for 13C NMR studies of the reactive site peptide bond Lys-15-Ala-16 in the complexes with [bovine] trypsin, trypsinogen and anhydrotrypsin. The chemical shift of [1-13C]Lys-15 was 175.7 ppm in the free inhibitor, 176.4 ppm in the complexes with trypsin and anhydrotrypsin and the ternary complex with trypsinogen and H-Ile-Val-OH and 175.7 ppm in a neutral solution containing the inhibitor and trypsinogen. These data show that the trypsin-BPTI complex does not contain a covalent tetrahedral carbon atom in the position of the reactive site peptide carbonyl of the inhibitor. They would be consistent with the formation of a noncovalent complex but cannot at present be used to further characterize the degree of a possible pyramidalization of the carbonyl carbon of Lys-15 in such a complex. The identical chemical shifts in the complexes with trypsin and anhydrotrypsin indicate that the .gamma.-hydroxyl group of Ser-195 of trypsin does not have an important role in the binding of the inhibitor. The previously described stepwise transition from the trypsinogen conformation to an intermediate conformational state in the trypsinogen-BPTI complex and a trypsin-like conformation in the ternary complex trypsinogen-BPTI-H-Ile-Val-OH appears to be manifested in the chemical shift of [1-13C]Lys-15 of labeled BPTI.