Glutamate carboxypeptidase III (GCPIII) is best known as a homologue of glutamate carboxypeptidase II [GCPII; also known as prostate-specific membrane antigen (PSMA)], a protease involved in neurological disorders and overexpressed in a number of solid cancers. However, mouse GCPIII was recently shown to cleave b-citrylglutamate (BCG), suggesting that these two closely related enzymes have distinct functions. To develop a tool to dissect, evaluate and quantify the activities of human GCPII and GCPIII, we analysed the catalytic efficiencies of these enzymes towards three physiological substrates. We observed a high efficiency of BCG cleavage by GCPIII but not GCPII. We also identified a strong modulation of GCPIII enzymatic activity by divalent cations, while we did not observe this effect for GCPII. Additionally, we used X-ray crystallography and computational modelling (quantum and molecular mechanical calculations) to describe the mechanism of BCG binding to the active sites of GCPII and GCPIII, respectively. Finally, we took advantage of the substantial differences in the enzymatic efficiencies of GCPII and GCPIII towards their substrates, using enzymatic assays for specific detection of these proteins in human tissues. Our findings suggest that GCPIII may not act merely as a complementary enzyme to GCPII, and it more likely possesses a specific physiological function related to BCG metabolism in the human body. DatabaseThe X-ray structure of GCPII Glu424Ala in complex with BCG has been deposited in the RCSB Protein Data Bank under accession code 5F09. Abbreviations 3D, three-dimensional; AAS, atomic absorption spectroscopy; ABS, arene-binding site; ACN, acetonitrile; BCG, b-citryl-L-glutamic acid (bcitrylglutamate, beta-citrylglutamate, beta-citryl-L-glutamic acid, beta-citryl-L-glutamate); BSA, bovine serum albumin; C12E8, octaethylene glycol monododecyl ether; DBU, 1,8-Diazabicycloundec-7-ene; DIEA, N,N-Diisopropylethylamine; EtOAc, ethylacetate; FolGlu n , folyl-n-c-Lglutamic acid; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; GCPII, glutamate carboxypeptidase II; GCPIII, glutamate carboxypeptidase III; HPLC, high-performance liquid chromatography; HRMS, high-resolution mass spectrometry; MeOH, methanol; MD/ MM, molecular dynamical/molecular mechanical calculations; NAAG, N-acetyl-L-aspartyl-L-glutamic acid; NMR, nuclear magnetic resonance; OPA, orthophtalaldehyde; ORF, open reading frame; Pd(C), palladium on activated charcoal; PSMA, prostate-specific membrane antigen; QM/MM, quantum mechanical and molecular mechanical calculations; QM/MM/MD, quantum mechanical, molecular mechanical and molecular dynamical calculations; qPCR, quantitative polymerase chain reaction; rhGCPII, recombinant human glutamate carboxypeptidase II; SDS/PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; TLC, thin-layer chromatography.
Virtually all low molecular weight inhibitors of human glutamate carboxypeptidase II (GCPII) are highly polar compounds that have limited use in settings where more lipophilic molecules are desired. Here we report the identification and characterization of GCPII inhibitors with enhanced liphophilicity that are derived from a series of newly identified dipeptidic GCPII substrates featuring non-polar aliphatic side chains at the C-terminus. To analyze the interactions governing the substrate recognition by GCPII, we determined crystal structures of the inactive GCPII(E424A) mutant in complex with selected dipeptides and complemented the structural data with quantum mechanics/molecular mechanics calculations. Results reveal the importance of non-polar interactions governing GCPII affinity towards novel substrates as well as formerly unnoticed plasticity of the S1′ specificity pocket. Based on those data, we designed, synthesized and evaluated a series of novel GCPII inhibitors with enhanced lipophilicity, with the best candidates having low nanomolar inhibition constants and clogD > -0.3. Our findings offer new insights into the design of more lipophilic inhibitors targeting GCPII.
In addition to its well-characterized role in the central nervous system, human glutamate carboxypeptidase II (GCPII, Uniprot ID Q04609) acts as a folate hydrolase in the small intestine, participating in the absorption of dietary polyglutamylated folates (FolGlun), which are the provitamin form of folic acid (also known as vitamin B9). Despite the role of GCPII as a folate hydrolase, nothing has been known about the processing of polyglutamylated folates by GCPII at the structural or enzymological level. Moreover, many epidemiologic studies on the relationship of the naturally occurring His475Tyr polymorphism to folic acid status suggest that this polymorphism may be associated with several pathologies linked to impaired folate metabolism. In this study we present 1) a series X-ray structures of complexes between a catalytically inactive GCPII mutant (Glu424Ala) and a panel of naturally occurring polyglutamylated folates, 2) X-ray structure of the His475Tyr variant at 1.83 Å resolution, 3) study of the recently identified arene-binding site of GCPII through mutagenesis (Arg463Leu, Arg511Leu, and Trp541Ala), inhibitor binding and enzyme kinetics with polyglutamylated folates as substrates and 4) comparison of the thermal stabilities and folate-hydrolyzing activities of GCPII wild type and His475Tyr variants. As a result, the crystallographic data reveal considerable details about the binding mode of polyglutamylated folates to GCPII, especially the engagement of the arene binding site in recognizing the folic acid moiety. Additionally, the combined structural and kinetic data suggest that GCPII wild type and His475Tyr variant are functionally identical.
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