Biomedical Engineering Reference
In-Depth Information
3.5.1.1 GCPIII: Closest Relative
Like the GCPII gene, the gene that encodes GCPIII is found on the 11th
chromosome, at position q14.3-q21. 17 Its exon/intron structure is very similar
to that of GCPII, resulting in a protein with 67% amino acid identity to GCPII.
GCPIII is a 740-amino-acid type II integral membrane protein.
The physiological role and protein localization of GCPIII are unknown.
Several studies show that human and mouse GCPIII is capable of NAAG
hydrolysis in vitro. 17,50,65 Bzdega et al. report that rat GCPII and mouse
GCPIII transfectants hydrolyze NAAG with a similar pH profile and perform
the hydrolysis with similar K M and V max values. 50 A more detailed study by
Hlouchova et al. later provided a direct comparison of purified human GCPII
and GCPIII and observed differences in the pH and salt dependence of the
peptidase activities as well as distinct substrate specificities of the two
enzymes. 65 Neither of these studies, however, confirmed any DPPIV activity of
GCPII or GCPIII, as suggested earlier by Pangalos and others. 17,50,65
A number of GCPII inhibitors have been used to monitor GCPIII inhibition,
and all of the inhibitors tested revealed that GCPII and GCPIII have similar
inhibitory profiles. 50,65,66 To date, no selective inhibitors of GCPII/GCPIII
have been designed.
3.6 Structure and Enzymology
3.6.1 Enzymology
3.6.1.1 Reaction Mechanism
The mechanism of substrate hydrolysis by GCPII is similar to other binuclear
metallopeptidases and was elucidated in detail by Klusak and co-workers (see
Scheme 3.1). 67 The concerted action of two active-site zinc ions together with
Glu424, which serves as a proton shuttle, is instrumental for peptide hydrolysis.
In the free enzyme, the two zinc ions are symmetrically bridged by a hydroxide
anion. Upon substrate (NAAG) binding, the scissile peptide bond is polarized
owing to the proximity of the peptide carbonyl to the catalytic Zn1 ion and its
interaction with the Tyr552 hydroxyl group. A proton extracted from the
bridged OH - by Glu424 is transported to the peptide bond nitrogen atom,
leading to the formation of a metastable tetrahedral intermediate. This first
proton transfer is followed by the second proton transfer from the OH - moiety,
already bound to the peptide bond carbon, to Glu424. At the same time, the
peptide bond is broken, and the reaction products (NAA and Glu) are released.
3.6.1.2 Substrate Specificity
Several studies have analyzed GCPII substrate specificity either by determining
hydrolysis of peptidic substrates directly or by inferring the enzyme's anity
for various peptides from inhibition data. 2,3,65,68-72 Major efforts have focused
on determining kinetic constants for two natural substrates—NAAG and
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