STRUCTURE OF THE COMPLEX OF L-BENZYLSUCCINATE WITH WHEAT SERINE CARBOXYPEPTIDASE-II AT 2.0-ANGSTROM RESOLUTION

The structure of the complex of L-benzylsuccinate (K-i = 0.2 mM) bound to wheat serine carboxypeptidase II has been analyzed at 2.0-Angstrom resolution for native and inhibited crystals at -170 degrees C. The m odel has been refined and has a standard crystallographic R-factor of 0.176 for 57 734 reflections observed between 20.0- and 2.0-Angstrom r esolution. The root mean square deviation from ideal bonds is 0.017 HL and from ideal angles is 2.6 degrees. The model consists of 400 amino acids, 4 N-linked saccharide residues, and 430 water molecules. L-Ben zylsuccinate occupies a narrow slot in the active site defined by Tyr 60, Tyr 239, and the polypeptide backbone. One carboxylate forms hydro gen bonds to Glu 145, Asn 51, the amide of Gly 52, and the catalytic H is 397, suggestive of how the peptide C-terminal carboxylate is recogn ized by the enzyme. The phenyl ring stacks between Tyr 239 and Tyr 60, while the other carboxylate occupies the ''oxyanion hole''. One of th e oxygens accepts hydrogen bonds from the amides of Tyr 147 and Gly 53 , while the other forms a very close contact (2.3 Angstrom) with the O gamma of Ser 146, forcing the side chain into a conformation alternat ive to that found in the resting state of the enzyme. The inhibitor oc cupies the active site in a way that suggests that it can be regarded as a transition-state analogue of serine carboxypeptidases. The model suggests a novel enzymatic mechanism, involving substrate-assisted cat alysis, that might account for the low pH optimum (4.0-5.5) of peptida se activity unique to this family of serine proteinases.