Glutamate Carboxypeptidase II as a Therapeutic Target - Proteinases as Drug Targets - page 75

3.6.2.1 Active Site and Specificity Pockets
The substrate-binding cavity of GCPII is composed of the active site (defined
by the presence of two zinc ions) flanked by the S1 0 and S1 specificity pockets.
The S1 0 pocket is defined as the part of a peptidase that harbors the P1 0 residue
of a cognate substrate, i.e. the residue C-terminally adjacent to the scissile
peptide bond. In the case of GCPII, the P1 0 residue is glutamate for any of the
naturally occurring substrates (NAAG or folate-poly-g-glutamates), but other
amino acids can be tolerated as well. The S1 pocket of GCPII (responsible for
the recognition of the P1 amino acid of a substrate) shows a strong preference
for negatively charged residues.
3.6.2.1.1 S1 0 Pocket (Pharmacophore Pocket). The S1 0 pocket of GCPII is
'optimized' for binding glutamate and glutamate-like moieties and is sometimes
referred to as the pharmacophore pocket. The pocket (1) confers selectivity, spe-
cificity, and anity towards GCPII-specific inhibitors; (2) interacts with the
majority of known low-molecular-weight GCPII inhibitors (excluding general
metalloenzyme inhibitors such as EDTA); and (3) is a 'docking module' for ori-
ginal GCPII inhibitors including quisqualate and 2-PMPA. 78,82 The S1 0 pocket
(having approximate dimensions of 0.8 0.8 0.8 nm) sits at the very bottom
of the substrate binding cavity and is shaped by amino acids Phe209, Arg210,
Asn257, Glu424, Glu425, Gly427, Leu428, Gly518, Lys699, and Tyr700.
3.6.2.1.2 Active Site. The GCPII active site, which harbors two zinc atoms
and the catalytic Glu424, separates the S1 and S1 0 specificity pockets. The
active site zincs are crucial for the design of high-anity GCPII inhibitors,
as documented by the nearly 10 6 -fold increase in anity from glutamate to
2-PMPA, its phosphonate analog (see Figure 3.2 for inhibitor structures).
Several zinc-interacting functionalities have been employed for inhibitor
design. The most prevalent classes of GCPII inhibitors today include thiols
and phosphorus-containing compounds such as phosphonates, phosphinates,
and phosphoamidates. 88 The latter, together with ureas, also serve as non-
hydrolyzable surrogates of the peptide bond in the inhibitor class derived
from NAAG (Section 3.7.1.2).
3.6.2.1.3 S1 Pocket (S1 Funnel). The S1 pocket of GCPII is fairly specific,
as only the presence of negatively charged residues (i.e. aspartate, glutamate,
and their analogs) at the P1 position allows for ecient substrate clea-
vage. 65,69 Such specificity is secured by the presence of the arginine patch—a
conspicuous spatial arrangement of the side chains of Arg534, Arg536, and
Arg463. The positively charged guanidinium groups of Arg534 and Arg536
interact directly with the P1 side chain of a substrate, providing a mechan-
istic explanation for the enzyme preference for acidic groups at the P1 posi-
tion. The architecture of the arginine patch is, at least in part, maintained by
the presence of the chloride anion, which holds Arg534 in an invariant