Refined atomic model of wheat serine carboxypeptidase II at 2.2-.ANG. resolution

Abstract

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-A 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 A and from bond angles of 2.8 degrees. The model supports the general conclusions of an earlier study at 3.5-A 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.(ABSTRACT TRUNCATED AT 400 WORDS)

An Expression and Bioinformatics Analysis of the Arabidopsis Serine Carboxypeptidase-Like Gene Family

“…Reverse transcription PCR results suggest that some SCPL genes are expressed in a highly tissue-specific fashion, whereas others are transcribed in a wide range of tissue types. Taken together, these data suggest that the Arabidopsis SCPL gene family encodes a diverse group of enzymes whose functions are likely to extend beyond protein degradation and processing to include activities such as the production of secondary metabolites.Serine carboxypeptidases (SCPs) are members of the a/b hydrolase family of proteins, which make use of a Ser-Asp-His catalytic triad to cleave the carboxyterminal peptide bonds of their protein or peptide substrates (Hayashi et al, 1973(Hayashi et al, , 1975Bech and Breddam, 1989;Liao and Remington, 1990;Liao et al, 1992;Ollis et al, 1992). Proteins that contain this catalytic triad and are otherwise homologous to SCPs have been found in a variety of organisms (Doi et al, 1980;Kim and Hayashi, 1983;Breddam, 1986;Baulcombe et al, 1987;Galjart et al, 1988;Bradley, 1992;Degan et al, 1994;Endrizzi et al, 1994;Wajant et al, 1994;Jones et al, 1996;Li and Steffens, 2000).…”

Organ-Specific and Hormone-Dependent Expression of Genes for Serine Carboxypeptidases during Development and Following Germination of Rice Grains

“…In spite of the divergence at both terminal sequences, type IH-like CPD has a hexapeptide that includes a Ser residue (G-E-S-Y-A-G, amino acid positions 146-151). This Ser residue, in association with His-393 and Asp-336, are applicable to the three key residues of wheat CPD II that form the 'catalytic triad' required for full activity of Ser CPDs (Galjart et al, 1990;Liao et al, 1992).…”

Crystallization of a soluble form of the Kexlp serine carboxypeptidase from Saccharomyces cerevisiae

“…The K , of Kexlp for dior tripeptide substrate analogues is typically about 0.5 mM, whereas that for a-factor-KR is 22 pM, and therefore the enzyme must be interacting with PI and other sites N-terminal to the hydrolyzed peptide bond; S1 subsites have not been firmly identified for either CPD-Y (Endrizzi et al, 1994) or CPDW-I1 (Liao et al, 1992). CPDW-I1 exists as a homodimer in solution, and the dimer interface is intriguing because it consists of three a-helices from one molecule packed against a P-sheet region of the second molecule, with the a-helices perpendicular, rather than parallel, to the strands of the sheet; in addition, the buried surface at the interface is unusual in that it is more hydrophilic than the rest of the molecular surface (Liao et al, 1992). Dimerization is also thought to be crucial to maintenance of the activity of human lysosomal protective protein (Zhou et al, 1991).…”

“…Despite the low level of sequence homology, the overall fold of CPD-Y is similar to that of CPDW-11, and large portions of the proteins, in particular the central &sheet, can be superimposed. Kexlp shares homology with these two enzymes over its entire sequence, and careful alignment using the structures of CPD-Y (Endrizzi et al, 1994) and CPDW-I1 (Liao et al, 1992) indicate that many of the differences occur in loop regions. Molecular replacement models have been constructed using this information and the search for a solution is underway.…”

“…From its homology with other carboxypeptidases, residues 23-506 define the catalytic domain of Kexlp. This domain shares 26% sequence identity with yeast serine carboxypeptidase (CPD-Y) and 22% sequence identity with wheat serine carboxypeptidase I1 (CPDW-II), two enzymes with known three-dimensional structures (Liao et al, 1992;Endrizzi et al, 1994). It also bears homology to other carboxypeptidases, among them a nonspecific serine carboxypeptidase from Aspergillus suitoi, originally thought to be an aspartic protease because of its enzymatic properties (Chiba et al, 1995); human lysosomal protective protein, a heterodimer consisting of 32-kDa and 20-kDa polypeptides held together by disulfide bridges, which is implicated in galactosialidosis (Galjart et al, 1988); and human prolylcarboxypeptidase (Tan et al, 1993), which is able to cleave Pro-X bonds, such as those found in angiotensin I1 and 111, and is expressed in a variety of human cell types.…”