Crystallization of serine carboxypeptidases

Wilson, Keith; Liao, D I; Bullock, T.L.; Remington, S.J.; Breddam, Klaus

doi:10.1016/0022-2836(90)90352-m

Cited by 8 publications

(5 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Crystallization. Wheat serine carboxypeptidase II (CPDW-II) was purified as previously described (Breddam et al, 1987;Wilson et al, 1990). Large single crystals were obtained by the hanging drop vapor diffusion method from polyethylene glycol/sodium chloride solutions as previously reported .…”

Section: Methodsmentioning

confidence: 99%

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

Liao¹,

Breddam²,

Sweet³

et al. 1992

Biochemistry

Self Cite

144

157

View full text Add to dashboard Cite

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)

show abstract

Section: Methodsmentioning

confidence: 99%

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

Liao¹,

Breddam²,

Sweet³

et al. 1992

Biochemistry

Self Cite

144

157

View full text Add to dashboard Cite

show abstract

“…Functional implications of these differences are not clear. Unlike the situation for serine endopeptidases, the importance ofthree domains for carboxypeptidase activity has not been established (34). Similar to all other serine carboxypeptidases in the family (27)(28)(29)(30)(31)(32), the mosquito VCP does not have any significant homology with any known sequences of trypsin-like serine endopeptidases or esterases.…”

mentioning

confidence: 95%

An extraovarian protein accumulated in mosquito oocytes is a carboxypeptidase activated in embryos.

Cho

Deitsch

Raikhel

1991

Proc. Natl. Acad. Sci. U.S.A.

102

View full text Add to dashboard Cite

We report a phenomenon previously unknown for oviparous animals; in Aedes aegypti mosquitoes a serine carboxypeptidase is synthesized extraovarially and then internalized by oocytes. The cDNA encoding mosquito vitellogenic carboxypeptidase (VCP) was cloned and sequenced. The VCP cDNA hybridizes to a 1.5-kilobase mRNA present only in the fat body of vitellogenic females. The deduced amino acid sequence of VCP shares significant homology with members of the serine carboxypeptidase family. Binding assays using a serine protease inhibitor, [3H~diisopropyl fluorophosphate, showed that VCP is activated in eggs at the onset of embryonic development. Activation of VCP is associated with the reduction in its size from 53 kDa (inactive proenzyme) to 48 kDa (active enzyme). The active, 48-kDa, form ofVCP is maximally present at the middle of embryonic development and disappears by the end.

show abstract

“…Deamidase also shares properties in common with cathepsin A, and as a serine carboxypeptidase, belongs to a family of enzymes together with yeast carboxypeptidase Y, KEX-1 gene product, 5 ' 7 plant carboxypeptidases, 9 and human prolylcarboxypeptidase. 10 The current and previous experiments 6 show that deamidase inactivates ET-1 by removing a single amino acid, Trp 21 .…”

Section: Discussionmentioning

confidence: 99%

Inactivation of endothelin-1 by an enzyme of the vascular endothelial cells.

et al. 1993

View full text Add to dashboard Cite

We previously investigated the inactivation of endothelin-1 by deamidase (lysosomal protective protein), present in many cells, including vascular smooth muscle cells. This enzyme, which we originally purified from human platelets, preferentially hydrolyzes peptides at the C-termlnus with hydrophobic amino adds in the Pi or P,' position or both and thereby inactivates endothelin-1, which has a C-tenninal sequence of Ile"-Ile

show abstract

Crystallization of serine carboxypeptidases

Cited by 8 publications

References 16 publications

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

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

An extraovarian protein accumulated in mosquito oocytes is a carboxypeptidase activated in embryos.

Inactivation of endothelin-1 by an enzyme of the vascular endothelial cells.

Contact Info

Product

Resources

About