Peptide Aldehyde Complexes with Wheat Serine Carboxypeptidase II: Implications for the Catalytic Mechanism and Substrate Specificity

Abstract

The structures of two ternary complexes of wheat serine carboxypeptidase II (CPD-WII), with a tetrapeptide aldehyde and a reaction product arginine, have been determined by X-ray crystallography at room temperature and −170°. The peptide aldehydes, antipain and chymostatin, form covalent adducts with the active-site serine 146. The CPD-WII antipain arginine model has a standard crystallographic R-factor of 0.162, with good geometry at 2.5 Å resolution for data collected at room temperature. The −170°C model of the chymostatin arginine complex has an R-factor of 0.174, with good geometry using data to 2.1 Å resolution. The structures suggest binding subsites N-terminal to the scissile bond. All four residues of chymostatin are well-localized in the putative S 1through S 4sites, while density is apparent only in S 1and S 2for antipain. In the S 1site, Val340 and 341, Phe215 and Leu216 form a hydrophobic binding surface, not a pocket, for the P 1phenylalanyl side-chain of chymostatin. The P 1arginyl of antipain also binds at this site, but the positive charge appears to be stabilized by additional solvent molecules. Thus, the hybrid nature of the S 1site accounts for the ability of CPD-WII to accept both hydrophobic and basic residues at P 1. Hydrogen bonds to the peptide substrate backbone are few and are made primarily with side-chains on the enzyme. Thus, substrate recognition by CPD-WII appears to have nothing in common with that of the other families of serine proteinases. The hemiacetal linkages to the essential Ser146 are of a single stereoisomer with tetrahedral geometry, with an oxygen atom occupying the "oxyanion hole" region of the enzyme. This atom accepts three hydrogen bonds, two from the polypeptide backbone and one from the positively-charged amino group of bound arginine, and must be negatively charged. Thus, the combination of ligands forms an excellent approxi mation to the oxyanion intermediate formed during peptide hydrolysis. Surprisingly, the (R) stereochemistry at the hemiacetal linkage is opposite to that expected by comparison to previously determined structures of peptide aldehydes complexed with Streptomyces griseusproteinase A. This is shown to be a consequence of the approximate mirror symmetry of the arrangement of catalytic groups in the two families of serine proteases and suggests that the stereochemical course of the two enzymatic reactions differ in handedness.