Reference "Refined atomic model of wheat serine carboxypeptidase II at 2.2-A resolution"

Reference ID

9077

Title

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

Source

Biochemistry (John Wiley & Sons), 1992, vol. 31, pp. 9796-9812

Authors (5)

Liao-D-I	Breddam-K
Sweet-R-M	Bullock-T
Remington-S-J

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)