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Reduced folic acid derivatives support biosynthesis of DNA, RNA and amino acids in bacteria as well as in eukaryotes, including humans. While the genes and steps for bacterial folic acid synthesis are known, those associated with folic acid catabolism are not well understood. A folate catabolite found in both humans and bacteria is p-aminobenzoyl-glutamate (PABA-GLU). The enzyme p-aminobenzoyl-glutamate hydrolase (PGH) breaks down PABA-GLU and is part of an apparent operon, the abg region, in E. coli. The subunits of PGH possess sequence and catalytic similarities to carboxypeptidase enzymes from Pseudomonas species. A comparison of the subunit sequences and activity of PGH, relative to carboxypeptidase enzymes, may lead to a better understanding of bacterial physiology and pathway evolution. We first compared the amino acid sequences of AbgA, AbgB and carboxypeptidase G2 from Pseudomonas sp. RS-16, which has been crystallized. Then we compared the enzyme activities of E. coli PGH and commercially available Pseudomonas carboxypeptidase G using spectrophotometric assays measuring cleavage of PABA-GLU, folate, aminopterin, methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate. The Km and Vmax values for the folate and anti-folate substrates of PGH could not be determined, because the instrument reached its limit before the enzyme was saturated. Therefore, activity of PGH was compared to the activity of CPG, or normalized to PABA-GLU (nmole/min/μg). Relative to its activity with 10 μM PABA-GLU (100%), PGH cleaved glutamate from methotrexate (48%), aminopterin (45%) and folate (9%). Reduced folates leucovorin (5-formyltetrahydrofolate) and 5-methyltetrahydrofolate were not cleaved by PGH. Our data suggest that E. coli PGH is specific for PABA-GLU as its activity with natural folates (folate, 5-methyltetrahydrofolate, and leucovorin) was very poor. It does, however, have some ability to cleave anti-folates which may have clinical applications in treatment of chemotherapy overdose.

Background: Carboxypeptidase G enzymes hydrolyze the C-terminal glutamate moiety from folic acid and its analogues, such as methotrexate. The enzyme studied here, carboxypeptidase G 2 (CPG 2 ), is a dimeric zinc-dependent exopeptidase produced by Pseudomonas sp. strain RS-16. CPG 2 has applications in cancer therapy: following its administration as an immunoconjugate, in which CPG 2 is linked to an antibody to a tumour-specific antigen, it can enzymatically convert subsequently administered inactive prodrugs to cytotoxic drugs selectively at the tumour site. CPG 2 has no significant amino acid sequence homology with proteins of known structure. Hence, structure determination of CPG 2 was undertaken to identify active-site residues, which may in turn provide ideas for protein and/or substrate modification with a view to improving its therapeutic usefulness. Results: We have determined the crystal structure of CPG 2 at 2.5 A resolution using multiple isomorphous replacement methods and non-crystallographic symmetry averaging. Each subunit of the molecular dimer consists of a larger catalytic domain containing two zinc ions at the active site, and a separate smaller domain that forms the dimer interface. The two active sites in the dimer are more than 60 A apart and are presumed to be independent; each contains a symmetric distribution of carboxylate and histidine ligands around two zinc ions which are 3.3 A apart. This distance is bridged by two shared zinc ligands, an aspartic acid residue and a hydroxyl ion. Conclusions: We find that the CPG 2 catalytic domain has structural homology with other zinc-dependent exopeptidases, both those with a single zinc ion and those with a pair of zinc ions in the active site. The closest structural homology is with the aminopeptidase from Aeromonas proteolytica , where the similarity includes superposable zinc ligands but does not extend to the rest of the active-site residues, consistent with the different substrate specificities. The mechanism of peptide cleavage is likely to be very similar in these two enzymes and may involve the bridging hydroxyl ion ligand acting as a primary nucleophile.

This chapter describes the structural chemistry and the biological aspects of carboxypeptidase G 3 . Carboxypeptidase G 3 consists of four subunits of identical molecular mass of 15 kDa. The pI is estimated to be 7.2. The enzyme is suspected to be a zinc-dependent metallopeptidase. The amino acid sequence and gene for carboxypeptidase G 3 are not known yet. The enzyme exists on the cell membrane of Pseudomonas sp. bacteria and seems to be a constitutive enzyme. The enzyme shows in vitro antitumor activity. Carboxypeptidase G 3 very slightly inhibits the growth of murine leukemia, but shows remarkable antitumor activities against human carcinoma KB and PC-3 cells among ten kinds of human carcinoma cell lines. Its antitumor activity mechanism seems to be based on the degradation of folic acid. Unlike carboxypeptidases G, G 1 and G 2 , the carboxypeptidase G 3 acts not only on the L-form of acidic amino acids, but also on the D-form and slightly hydrolyzes Bz-DL-Glu. However, some properties such as C-terminal glutamate-releasing activity and the requirement for Zn 2+ are quite similar to those of the other enzymes.

Publisher Summary This chapter describes the activity, specificity and structural chemistry of glutamate carboxypeptidase. Glutamate carboxypeptidases are characterized as Zn2+ requiring exopeptidases with specificity for glutamate and this specificity is not absolute as carboxypeptidase G was reported as exhibiting activity against pteroyl compounds with C-terminal aspartic acid. Physicochemical properties and substrate affinities for folic acid, and a variety of analogs are compared for the G1 and G2 enzymes. Carboxypeptidase G2 is a dimeric protein of 83,600 kDa containing two atoms of zinc per subunit. There are no disulfide bonds and the complete nucleotide and amino acid sequences were resolved following cloning of the gene into Escherichia coli. In both native and recombinant form the enzyme is located in the periplasmic space, targeted by a 22 amino acid signal peptide. Carboxypeptidase G2 has been crystallized and the crystal structure determined at 2.5 A resolutions. Each subunit of the molecular dimer consists of a larger catalytic domain containing two zinc ions at the active site and a separate smaller domain that forms the dimer interface.

Carboxypeptidase G(2) (CPG(2)) is a zinc-metalloenzyme employed in a range of cancer chemotherapy strategies by activating selectively nontoxic prodrugs into cytotoxic drugs in tumor as well as in the treatment of intoxication caused by high-doses of the anticancer drug methotrexate (MTX). CPG(2) catalyzes the hydrolytic cleavage of C-terminal of glutamate moiety from folic acid and analogues. Regardless of its extensive application, its mechanism of catalysis has not yet been determined and, so far, no co-crystallized complex has been published. So, in this study, molecular docking and a short molecular dynamics (MD) simulation sampling scheme, as a function of temperature, were performed to investigate a possible binding mode for MTX, a recognized substrate of CPG(2). The findings suggested that MTX interacts possibly in quite specific points of the CPG(2) active site, which are probably responsible for the molecular recognition and cleavage procedures. The MTX substrate fits well in the catalytic site by accommodating the pteridine moiety in an adjacent pocket to the active site whereas a glutamate moiety is pointed toward the protein surface. Additionally, a glutamate residue can interact with a crystallization water molecule in the active site, supporting its activation as a nucleophilic group.

Conjugates of F(ab′) 2 fragment of the monoclonal antibody A5B7 coupled to carboxypeptidase G 2 (CPG 2 ) have been produced using the heterobifunctional reagents 2-mercapto-[ S -acetyl]acetic acid, N -hydroxysuccinimide ester (SATA) and m -maleimidobenzoyl- N -hydroxysuccinimide ester (SMPB). The effect of various levels of modifying reagent on enzyme activity and antigen binding activity were determined, and it was shown that whilst CPG 2 is relatively sensitive to modification, insertion of three maleimide groups per CPG 2 resulted in the loss of 30% of enzyme activity; 5B7 F(ab′) 2 was insensitive to modification, little or no activity being lost. The coupling efficiency of the reation was shown to be fairly constant over a wide range of substitution levels. There was thus no advantage to be gained in using high substitution levels, which may result in loss of enzyme activity. The formation of undesired high molecular weight aggregates could be controlled by adjustment of the protein concentration during the final coupling step.

The amino acid sequence analysis of the human and porcine aminoacylases-1, the carboxypeptidase S precursor from Saccharomyces cerevisiae, the succinyl-diaminopimelate desuccinylase from Escherichia coli, Haemophilus influenzae and Corynebacterium glutamicum, the acetylornithine deacetylase from Escherichia coli and Dictyostelium discoideum and the carboxypeptidase G(2) precursor from Pseudomonas strain, using the Basic Local Alignment Search Tool (BLAST) and the Position-Specific Iterated BLAST (PSI-BLAST), allowed us to suggest that all these enzymes, which share common functional and biochemical features, belong to the same structural family. The three amino acid blocks which were found to be highly conserved, using the CLUSTAL W program, could be assigned to the catalytic active site, based on the general three-dimensional structure of the carboxypeptidase G(2) from the Pseudomonas strain precursor. Six additional proteins with the same signature have been retrieved after performing two successive PSI-BLAST iterations using the sequence of the conserved motif, namely Lactobacillus delbrueckii aminoacyl-histidine dipeptidase, Streptomyces griseus aminopeptidase, Saccharomyces cerevisiae aminopeptidase Y precursor, two Bacillus stearothermophilus N-carbamyl-L-amino acid amidohydrolases and Pseudomonas sp. hydantoin utilization protein C. The three conserved amino acid motifs corresponded to the following blocks: (i) [S, G, A]-H-x-D-x-V; (ii) G-x-x-D; and (iii) x-E-E. This new sequence signature is clearly different from that commonly reported in the literature for proteins belonging to the ArgE/DapE/CPG2/YscS family.

Several N-thiophosphonyl-glutamates were found to be potent competitive inhibitors of a zinc-dependent glutamyl hydrolase, carboxypeptidase G (CPG). Weak inhibition exhibited by an analogous N-phosphonyl-glutamate suggests that the enhanced potency of the phosphonamidothioates is due to the presence of their sulfur ligand and its favorable interactions with active site features, presumably zinc(II).

The carboxypeptidase G class of enzymes rapidly hydrolyze methotrexate (MTX) into the inactive metabolites 4-deoxy-4-amino-N10-methylpteroic acid (DAMPA) and glutamate. This study evaluated the use of carboxypeptidase-G2 (CPDG2) as a potential intrathecal (IT) rescue agent for massive IT MTX overdose. The CSF pharmacokinetics of MTX with and without CPDG2 rescue was studied in adult rhesus monkeys (Macaca mulatta) using a nontoxic IT 5 mg dose (equivalent to 50 mg in humans). Without CPDG2 rescue, peak CSF MTX concentration was 2,904 +/- 340 mumol/L. Within 5 minutes of administration of 30 U IT CPDG2, CSF MTX concentrations decreased greater than 400-fold to 6.55 +/- 6.7 microM. Subsequently, groups of three monkeys received either 25 mg IT MTX (equivalent to 250 mg in humans) followed by 150 U IT CPDG2 or 50 mg IT MTX (equivalent to 500 mg in humans) followed by 300 U IT CPDG2. All animals survived without neurotoxicity. Our studies suggest that CPDG2 may prove to be an important addition to the currently recommended strategy for the management of IT MTX overdose.

Conjugation of carboxypeptidase G and arginase, two enzymes of therapeutic interest, to a soluble dextran significantly enhanced plasma persistence in normal and tumour-bearing mice. A prolonged decrease in arginine concentrations in plasma of tumour-bearing mice was demonstrated by using the dextran-linked arginase. Gel filtration of dextran-enzyme conjugate showed that enzyme activity co-chromatographed as a single peak with carbohydrate, and enzyme was shown to be covalently linked to the dextran.

1. 1. Four distinct carboxypeptidases (EC 3.4.12.-) were shown to exist in rat liver lysosomes: cathepsin A, catheptic carboxypeptidase B, catheptic carboxypeptidase C, and catheptic carboxypeptidase G. These carboxypeptidases were distinguished on the basis of substrate specificity, thiol requirement, and separation by Sephadex G-100 column chromatography. The K m and pH optimum for each carboxypeptidase were determined. 2. 2. From the Sephadex G-100 elution profile, two peaks of cathepsin A activity were found. The major cathepsin A peak, cathepsin A1, was further purified by ion-exchange chromatography on DEAE-cellulose. 3. 3. The highly purified cathepsin A1 fraction hydrolyzed N- benzyloxycarbonyl (Cbz)-Glu-Phe, Cbz-Gly-Phe, and Ac-Phe-Tyr, which suggests that these hydrolytic activities were due to the same enzyme. 4. 4. Catheptic carboxypeptidase B and catheptic carboxypeptidase G eluted from the Sephadex G-100 column along with cathepsin B2. This suggests that cathepsin B2 may have carboxypeptidase activity. 5. 5. The purification of the lysosomal carboxypeptidases over homogenate activities was: cathepsin A1, 1280-fold; catheptic carboxypeptidase B, 220-fold; and catheptic carboxypeptidase G, 920-fold. Catheptic carboxypeptidase C was purified 16-fold over light-mitochondrial supernatant activity.

The extraordinary specificity of antigen-antibody reactions forms the basis of a simplified isolation method for antigens. The use of antigens to isolate antibodies has been employed by immunologists for many years [1, 2]. The early use of imnunoadsorbents to isolate antibodies was not always successful because the physical adsorption used to fix the antigen permitted “leaking”. However, with the various methods available today for covalently bonding to solid supports, this is no longer a serious problem.

An enzyme, carboxypeptidase G1, extracted from a strain of Pseudomonas stutzeri, has been isolated which hydrolyzes the carboxyl-terminal glutamate from both reduced and nonreduced folate derivatives, with Km values between 1.1 and 18.1 x 10-6 m. This enzyme also hydrolyzes the COOH-terminal glutamate of oligopeptides and N-benzyloxycarbonyl glutamates; it demonstrates lesser activity against aspartate COOH-terminal peptide linkages, with Km values of 1 to 5 x 10-4 m. The reverse reaction (pteroate + glutamate → folate) is also catalyzed, but at an equilibrium lying far toward hydrolysis. The enzyme has been purified 1050-fold. It has a broad pH optimum over the range of 6.3 to 7.3 and has a molecular weight of 92,000 as estimated by calibrated Sephadex gel filtration. On disc gel electrophoresis in 1% sodium dodecyl sulfate, purified preparations migrate as a single band of 46,000 molecular weight, indicating that the enzyme is a dimeric protein. The enzyme is activated by Zn++. Enzymic cleavage of 4-amino-N10-methylpteroylglutamic acid (methotrexate) was strongly inhibited by the product l-glutamic acid, and to a lesser extent by l-aspartic acid, while d-glutamic acid was ineffective. Competitive inhibition of methotrexate cleavage was also demonstrated by the substrates 4-aminopteroylaspartic acid, N-benzyloxycarbonyl glutamic acid, and N-benzyloxycarbonyl aspartic acid. Although certain properties of carboxypeptidase G1 were similar to those from other sources, several differences were noted between this enzyme and previously described carboxypeptidases.

Several reactive azoic dichlorotriazinyl dyes specifically and irreversibly inactivate the folate-degrading enzyme carboxypeptidase G-2 at a site competitive with the enzyme substrates methotrexate (4-amino-N10-methylfolic acid) and p-aminobenzoyl-L-glutamate. Although the less reactive monochlorotriazinyl dye, Procion red H-8BN, is unable to inactivate the enzyme, it is capable of marked inhibition of inactivation by dichlorotriazinyl dyes in the presence of Zn2+. Zinc ions and, to a lesser extent other first row transition metal ions, significantly enhance the affinity of Procion red H-8BN and its analogues Procion red MX-8B and Procion red MX-2B, for carboxypeptidase G-2. It is proposed that this effect is mediated through the formation of a specific tetracoordinate Zn2+ complex between the azo linkage and adjacent sulphonate and hydroxyl functions of the dye and an appropriate ligand on the protein. Carboxypeptidase G-2 quantitatively inactivated with the dichlorotriazinyl dye, Procion red MX-8B, contains approximately 1 mol dye/mol subunit of Mr 42000. Proteolytic cleavage of the labelled enzyme and resolution of the peptides by reverse-phase high-performance liquid chromatography yields a principal red peptide which on amino acid sequence analysis results in the identification of the dye binding domain. The affinity label, Procion red MX-8B, is believed to be attached to the hydroxylic side chain of Thr-279.

A folate-degrading enzyme, carboxypeptidase G2, has been purified on a large scale from Pseudomonas sp. strain RS-16. Homogeneous enzyme was obtained by a three-step procedure involving ion-exchange chromatography and a novel triazine dye (affinity) chromatography step which utilizes Zn2+ to promote adsorption of the enzyme. Enzyme was selectively eluted by the use of a chelating agent (EDTA) and a step change in pH. The enzyme is a dimeric protein (Mr 83000) with two identical subunits of 41800 and contains four atoms of zinc per enzyme molecule, which are required for full activity. The enzyme follows Michaelis-Menten kinetics with Km values of 4.0 microM for folate, 8.0 microM for methotrexate and 34.0 microM for 5-methyltetrahydrofolate, the predominant form of reduced folate found in plasma.