STRUCTURE OF A SOLUBLE FORM OF THE KEX1P SERINE CARBOXYPEPTIDASE FROM SACCHAROMYCES CEREVISIAE. Brian H. Shilton, Yunge Li, Daniel Tessier, David Y. Thomas and Miroslaw Cygler Montreal Joint Centre for Structural Biology Biotechnology Research Institute, NRC, 6100 Royalmount Avenue Montreal, Quebec H4P 2R2

Kex1p, a prohormone processing enzyme from Saccharomyces cerevisiae, is a membrane-anchored serine carboxypeptidase involved in the maturation of alpha-pheromone and K1 killer toxin. Residues 23 to 506 represent the catalytic domain of Kex1p which exhibits sequence similarity to a number of other carboxypeptidases, including three for which crystal structures have been solved-wheat serine carboxypeptidase (CPDW-II; Liao et al., 1992, Biochemistry 31:9796-9812), yeast carboxypeptidase Y (CPD-Y; Endrizzi et al., 1994, Biochemistry 33:11106-11120), and human protective protein (Rudenko et al., 1995, Structure 3:1249-1259). Kex1p is unique among these proteins in that it is highly specific for lysyl or arginyl residues at the C-terminus of the substrate. We are interested in the functional differences between Kex1p and related carboxypeptidases, as well as the relationship between these enzymes and the larger alpha/beta hydrolase family.

Expression of a genetic construct coding for the catalytic domain of Kex1p yielded a soluble form of the enzyme (Kex1p-s; Mr = 56 kDa; Latchinian-Sadek and Thomas, 1994, Eur. J. Biochem, 219:647-652) which retains the kinetic characteristics of its membrane-bound parent. Kex1p-s has been crystallized in space group P2₁2₁2₁ with unit cell dimensions a=56.6 Å, b=84.0 Å, c=111.8 Å (Shilton et al., 1996, Protein Science, in the press); there is one molecule in the asymmetric unit. The structure has been solved by a combination of MIR and molecular replacement, and is currently under refinement against a 2.4 Å native data set collected at 120 K.