PEPTIDE ALDEHYDE COMPLEXES WITH WHEAT SERINE CARBOXYPEPTIDASE-II - IMPLICATIONS FOR THE CATALYTIC MECHANISM AND SUBSTRATE-SPECIFICITY

The structures of two ternary complexes of wheat serine carboxypeptida se II (CPD-WII), with a tetrapeptide aldehyde and a reaction product a rginine, have been determined by X-ray crystallography at room tempera ture and -170 degrees. The peptide aldehydes, antipain and chymostatin , form covalent adducts with the active-site serine 146. The CPD-WII a ntipain arginine model has a standard crystallographic R-factor of 0.1 62, with good geometry at 2.5 Angstrom resolution for data collected a t room temperature, The -170 degrees C model of the chymostatin argini ne complex has an R-factor of 0.174, with good geometry using data to 2.1 Angstrom resolution. The structures suggest binding subsites N-ter minal to the scissile bond. All four residues of chymostatin are well- localized in the putative S-1 through S-1 sites, while density is appa rent only in S-1 and S-2 for antipain. In the S-1 site, Val340 and 341 , Phe215 and Leu216 form a hydrophobic binding surface, not a pocket, for the P-1 phenylalanyl side-chain of chymostatin. The P-1 arginyl 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-1 site accounts for the ability oi: CPD-WII to accept both h ydrophobic and basic residues at pi. Hydrogen bonds to the peptide sub strate backbone are few and are made primarily with side-chains on the enzyme. Thus, substrate recognition by CPD-WII appears to have nothin g in common with that of the other families of serine proteinases. The hemiacetal linkages to the essential Ser146 are of a single stereoiso mer with tetrahedral geometry, with an oxygen atom occupying the ''oxy anion hole'' region of the enzyme. This atom accepts three hydrogen bo nds, two from the polypeptide backbone and one from the positively-cha rged amino group of bound arginine, and must be negatively charged. Th us, the combination of ligands forms an excellent approximation 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 griseus proteinase A. This is s hown to be a consequence of the approximate mirror symmetry of the arr angement of catalytic groups in the two families of serine proteases a nd suggests that the stereochemical course of the two enzymatic reacti ons differ in handedness. (C) 1996 Academic Press Limited.