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Human Glutamate Carboxypeptidase II (human + glutamate_carboxypeptidase_ii)
Selected AbstractsMapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesisFEBS JOURNAL, Issue 18 2007Petra Ml, ochová Human glutamate carboxypeptidase II [GCPII (EC 3.4.17.21)] is recognized as a promising pharmacological target for the treatment and imaging of various pathologies, including neurological disorders and prostate cancer. Recently reported crystal structures of GCPII provide structural insight into the organization of the substrate binding cavity and highlight residues implicated in substrate/inhibitor binding in the S1, site of the enzyme. To complement and extend the structural studies, we constructed a model of GCPII in complex with its substrate, N -acetyl- l -aspartyl- l -glutamate, which enabled us to predict additional amino acid residues interacting with the bound substrate, and used site-directed mutagenesis to assess the contribution of individual residues for substrate/inhibitor binding and enzymatic activity of GCPII. We prepared and characterized 12 GCPII mutants targeting the amino acids in the vicinity of substrate/inhibitor binding pockets. The experimental results, together with the molecular modeling, suggest that the amino acid residues delineating the S1, pocket of the enzyme (namely Arg210) contribute primarily to the high affinity binding of GCPII substrates/inhibitors, whereas the residues forming the S1 pocket might be more important for the ,fine-tuning' of GCPII substrate specificity. [source] Biochemical characterization of human glutamate carboxypeptidase IIIJOURNAL OF NEUROCHEMISTRY, Issue 3 2007Klára Hlouchová Abstract Human glutamate carboxypeptidase II (GCPII) is a transmembrane metallopeptidase found mainly in the brain, small intestine, and prostate. In the brain, it cleaves N -acetyl- l -aspartyl-glutamate, liberating free glutamate. Inhibition of GCPII has been shown to be neuroprotective in models of stroke and other neurodegenerations. In prostate, it is known as prostate-specific membrane antigen, a cancer marker. Recently, human glutamate carboxypeptidase III (GCPIII), a GCPII homolog with 67% amino acid identity, was cloned. While GCPII is recognized as an important pharmaceutical target, no biochemical study of human GCPIII is available at present. Here, we report the cloning, expression, and characterization of recombinant human GCPIII. We show that GCPIII lacks dipeptidylpeptidase IV-like activity, its activity is dependent on N -glycosylation, and it is effectively inhibited by several known inhibitors of GCPII. In comparison to GCPII, GCPIII has lower N -acetyl- l -aspartyl-glutamate-hydrolyzing activity, different pH and salt concentration dependence, and distinct substrate specificity, indicating that these homologs might play different biological roles. Based on a molecular model, we provide interpretation of the distinct substrate specificity of both enzymes, and examine the amino acid residues responsible for the differences by site-directed mutagenesis. These results may help to design potent and selective inhibitors of both enzymes. [source] A high-resolution structure of ligand-free human glutamate carboxypeptidase IIACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2007Cyril Barinka Human glutamate carboxypeptidase II (GCPII; EC 3.4.17.21) is an established marker for prostate-cancer diagnosis as well as a candidate therapeutic target for the treatment of diverse pathologies that involve glutamatergic transmission. Structural data on GCPII are thus valuable for the design and optimization of GCPII-specific inhibitors and diagnostic probes. The currently available structure of ligand-free GCPII was refined to a resolution of 3.5,Ĺ. This work reports the structure of the protein refined to 1.65,Ĺ resolution, with crystallographic values of R = 0.207 and Rfree = 0.228. The new structure extends the resolution appreciably and the new model based on this data shows significant differences when compared with the previously published model. [source] | ||||||||||