Pig pancreatic anhydro-elastase. Role of the serine-195 hydroxy group in the binding of inhibitors and substrate.

The binding constants of a number of ligands were measured for pancreatic elastase (PE) and anhydro-elastase (AE) in order to assess the contribution of Ser-195 to substrate and inhibitor binding by PE. AE was purified by affinity chromatography on a column containing immobilized turkey ovomucoid inhibitor. The AE had 0.1 +/- 0.1% of the activity of the native enzyme and contained 0.8 +/- 0.06 residue of dehydroalanine per molecule. A difference electron-density map, derived from an X-ray crystallographic analysis of AE, showed that the modified residue was Ser-195. The complexing of 3-carboxypropionyl-Ala-Ala-Ala-p-nitroanilide (SAN) to the active site of AE was also demonstrated by X-ray-diffraction analysis of an AE crystal soaked overnight with substrate. The nitroanilide moiety was not observed in the difference map. AE was shown to bind turkey ovomucoid inhibitor with a dissociation constant (Kd) of 0.3 +/- 0.06 microM compared with 0.10 microM for PE. The Kd of the AE-SAN complex (0.2 mM) was comparable with the Michaelis constant for SAN with PE (1.0 mM). A number of inhibitors, such as elastatinal, which forms a hemiketal adduct with PE, while others such as the beta-lactams, which function as acylators of the active-site serine residue, bound AE with a lower affinity than to PE. The binding of a peptidylchloromethane (acetyl-Ala-Ala-Pro-Ala-CH2Cl) to AE occurs without evidence for alkylation of histidine. The binding constants for benzoisothiazolinone and 3,4-dichloroisocoumarin to PE differed from their binding constants to AE by less than a factor of 4.0-fold. The contribution of the hydroxy group of Ser-195 to the binding of these inhibitors to PE in their non-covalent complexes is relatively small, even though they inactivate PE by an acylation mechanism. These results suggest that the hydroxy group on Ser-195 in PE is of secondary importance in the energetics of ligand binding, in contrast with its essential role in the catalytic properties of the enzyme.