REFINED ATOMIC MODEL OF WHEAT SERINE CARBOXYPEPTIDASE-II AT 2.2-ANGSTROM RESOLUTION

The crystal structure of the homodimeric serine carboxypeptidase II from wheat (CPDW-II, M(r) 120K) has been determined and fully refined at 2.2-angstrom resolution to a standard crystallographic R factor of 16.9% using synchrotron data collected at the Brookhaven National Laboratory. The model has an rms deviation from ideal bond lengths of 0.018 angstrom and from bond angles of 2.8-degrees. The model supports the general conclusions of an earlier study at 3.5-angstrom resolution and will form the basis for investigation into substrate binding and mechanistic studies. The enzyme has an alpha+beta fold, consisting of a central 11-stranded beta-sheet with a total of 15 helices on either side. The enzyme, like other serine proteinases, contains a "catalytic triad' Ser146-His397-Asp338 and a presumed "oxyanion hole" consisting of the backbone amides of Tyr147 and Gly53. The carboxylate of Asp338 and imidazole of His397 are not coplanar in contrast to the other serine proteinases. A comparison of the active site features of the three families of serine proteinases suggests that the "catalytic triad" should actually be regarded as two diads, a His-Asp diad and a His-Ser diad, and that the relative orientation of one diad with respect to the other is not particularly important. Four active site residues (52, 53, 65, and 146) have unfavorable backbone conformations but have well-defined electron density, suggesting that there is some strain in the active site region. The binding of the free amino acid arginine has been analyzed by difference Fourier methods, locating the binding site for the C-terminal carboxylate of the leaving group. The carboxylate makes hydrogen bonds to Glu145, Asn51, and the amide of Gly52. The carboxylate of Glu145 also makes a hydrogen bond with that of Glu65, suggesting that one or both may be protonated. Thus, the loss of peptidase activity at pH > 7 may in part be due to deprotonation of Glu145. The active site does not reveal exposed peptide amides and carbonyl oxygen atoms that could interact with substrate in an extended beta-sheet fashion. The fold of the polypeptide backbone is completely different than that of trypsin or subtilisin, suggesting that this is a third example of convergent molecular evolution to a common enzymatic activity, Furthermore, it is suggested that the active site sequence motif "G-X-S-X-G/A", often considered the hallmark of serine peptidase or esterase activity, is fortuitous and not the result of divergent evolution. However, the fold of CPDW-II has a remarkable similarity to four other classes of hydrolytic enzymes for which representative structures have recently been determined. These are the acetylcholine esterase, several triacylglycerol lipases, haloalkane dehalogenase, and dienelactone hydrolase. Thus, serine carboxypeptidases are suggested to illustrate the process of both convergent and divergent molecular evolution. A comparison of the amino acid sequences of human serine carboxypeptidase carbL, or "protective protein", and CPDW-II suggests that the dimer of CPDW-II may be a reasonable model for the dimer of the human enzyme and that the dimer interface is similar in the two proteins. This may be useful toward the understanding of human genetic disorders such as galactosialidosis, which can result from lesions in this locus [Zhou, X. Y., Galjart, N. J., Willemsen, R., Gillemans, N., Galjaard, H., & d'Azzo, A. (1991) EMBO J. 10, 4041-4048].