The X-ray crystal structure of bovine (gamma symbol)-carboxypeptidase A

Abstract

The structure of [gamma]-carboxypeptidase A from bovine pancreas has been determined by single-crystal X-ray crystallography at 2.0 [angstrom] with a final Residual R factor of 0.250. Multiple isomorphous replacement, an entropy-maximization procedure, and least squares refinement were used to determine the structure. The molecule has a zinc atom at the active site which is coordinated to His 69, His 196, and Glu 72. The enzyme crystallizes in space group P2[1] with unit cell parameters: a = 51.35(3) [angstrom], b = 57.77(2) [angstrom], c = 45.33(3) [angstrom], β = 94.85(6)°, V = 133,990 [angstrom]^3. There are two molecules per unit cell. The structure of CPA[gamma] is more diffuse than CPA[α]; it is approximately 15 to 20% larger. The hydrogen bonding which is present within the molecule is weaker, mostly as a result of the nearby segments of polypeptide backbone having been spread farther apart. Three cis-peptides are present in CPA[gamma] and occur between Ser 197 and Tyr 198, Pro 205 and Tyr 206, and Arg 272 and Asp 273. All these are immediately adjacent to catalytically important residues. The Tyr 248 -OH group is in the "up" position as would be expected for a non-substrate-bound structure (free enzyme.) Its distance from the active site appears greater than could be overcome by simple movements of the polypeptide backbone. Glu 270 appears near the active site. The carboxylate oxygens are 4.6 and 4.2 [angstrom] away from zinc. The elements of primary, secondary, and tertiary structure are presented and discussed. A list of refined atomic parameters is presented in Appendix B.