Molecular modeling and site-directed mutagenesis define the catalytic motif in human gamma -glutamyl hydrolase.

Human gamma-glutamyl hydrolase (hGH) is a central enzyme in folyl and antifolylpoly-gamma-glutamate metabolism, which functions by catalyzing the cleavage of the gamma-glutamyl chain of substrates. We previously reported that Cys-110 is essential for activity. Using the sequence of hGH as a query, alignment searches of protein data bases were made using the SSearch and TPROBE programs. Significant similarity was found between hGH and the glutamine amidotransferase type I domain of Escherichia coli carbamoyl phosphate synthetase. The resulting hypothesis is that the catalytic fold of hGH is similar to the folding of this domain in carbamoyl phosphate synthetase. This model predicts that Cys-110 of hGH is the active site nucleophile and forms a catalytic triad with residues His-220 and Glu-222. The hGH mutants C110A, H220A, and E222A were prepared. Consistent with the model, mutants C110A and H220A were inactive. However, the V(max) of the E222A hGH mutant was reduced only 6-fold relative to the wild-type enzyme. The model also predicted that His-171 in hGH may be involved in substrate binding. The H171N hGH mutant was found to have a 250-fold reduced V(max). These studies to determine the catalytic mechanism begin to define the three dimensional interactions of hGH with poly-gamma-glutamate substrates.

Glutamyl hydrolase. pharmacological role and enzymatic characterization.

gamma-Glutamyl hydrolase (GH, EC 3.4.19.9) is a lysosomal and secreted glycoprotein that hydrolyzes the gamma-glutamyl tail of antifolate and folate polyglutamates. Tumor cells that have high levels of GH are inherently resistant to classical antifolates, and further resistance can be acquired by elevations in GH following exposure to this class of antitumor agents. The highest level of expression in normal tissues occurs in the liver and kidney in humans. When panels of tumors are compared with normal tissues, GH expression is elevated in cancerous hepatic and breast tissue. A second poly-gamma-glutamate hydrolyzing enzyme, glutamate carboxypeptidase II, is a transmembrane protein whose active site is on the outside of the cell, occurring in the prostate gland, small intestine, brain, kidney, and tumor neovasculature. It is a high-affinity (nanomolar), low-turnover, zinc co-catalytic enzyme. In contrast, GH is a low-affinity (micromolar), high-turnover enzyme that has a cysteine at the active site. Data are presented suggesting that Cys110 is the nucleophile that attacks the gamma-amide linkage and causes hydrolysis. GH is being evaluated as an intracellular target for inhibition in order to enhance the therapeutic activity of antifolates and fluorouracil.

Glutamyl hydrolase: properties and pharmacologic impact.

Glutamyl hydrolase cleaves the poly-gamma-glutamate chain folate and antifolate poly-gamma-glutamates. Its cellular location is lysosomal with large amounts of the enzyme constitutively secreted. The highest levels of glutamyl hydrolase mRNA in humans is found in the liver and kidney. Baculovirus-expressed human enzyme has been used to evaluate the method of hydrolysis of methotrexate-gamma-glu4 and MTA-gamma-glu4. In both cases, the substrates are hydrolyzed by removal of the outer two gamma-glutamate linkages, yielding glu and gamma-glu2 as the glutamate products. Cell lines resistant to 5,10-dideazatetrahydrofolate (lometrexol) have sevenfold higher activities of glutamyl hydrolase. These cultures have a 60% to 90% reduced amount of antifolate polygamma-glutamates and 30% reduced folyl poly-gamma-glutamates. These results suggest the possibility of using glutamyl hydrolase to favorably modulate the activity of antifolate therapy.

Structural organization of the human gamma-glutamyl hydrolase gene.

Gamma-glutamyl hydrolase (GH) plays an important role in the metabolism of folic acid and the pharmacology of antifolates such as methotrexate. We have previously cloned and characterized the human GH cDNA. In this report, the complete organization and structure of the human GH gene was determined. The human GH gene spans 24 kb in the human genome, with nine exons sized from 51 to 371 bp. All of exon-intron splice junctions follow the GT-AG rule. The sequence upstream of exon 1 consists of a promoter-like, GC-rich region and a number of putative cis active elements including Sp1, AP1, and MZF1 sites. A TATA sequence in the 5' region of human GH gene was not observed, similar to housekeeping genes known to be tissue-specific and differentially expressed. S1 nuclease protection analysis with human liver, prostate, brain, and mammary gland revealed a major transcription start point at nucleotide -125 relative to the ATG start codon and several minor transcription start points. Analysis of GH cDNA isolated from human liver indicated a nucleotide change, T-->C, in the leader sequence of GH, which suggested a polymorphism. Studies of cDNA from different human tissue sources provided evidence that there is a single spliced cDNA species in human.

Site-directed mutagenesis establishes cysteine-110 as essential for enzyme activity in human gamma-glutamyl hydrolase.

Gamma-glutamyl hydrolase (GH), which hydrolyses the gamma-glutamyl conjugates of folic acid, is a key enzyme in the maintenance of cellular folylpolyglutamate concentrations. The catalytic mechanism of GH is not known. Consistent with earlier reports that GH is sulphydryl-sensitive, we found that recombinant human GH is inhibited by iodoacetic acid, suggesting that at least one cysteine is important for activity [Rhee, Lindau-Shepard, Chave, Galivan and Ryan (1998) Mol. Pharmacol. 53, 1040-1046]. Using site-directed mutagenesis, the cDNA for human GH was altered to encode four different proteins each with one of four cysteine residues changed to alanine. Three of the mutant proteins had activities similar to wild-type GH and were inhibited by iodoacetic acid, whereas the C110A mutant had no activity. Cys-110 is conserved among the human, rat and mouse GH amino acid sequences. The wild-type protein and all four mutants had similar intrinsic fluorescence spectra, indicating no major structural changes had been introduced. These results indicate that Cys-110 is essential for enzyme activity and suggest that GH is a cysteine peptidase. These studies represent the first identification of the essential Cys residue in this enzyme and provide the beginning of a framework to determine the catalytic mechanism, important in defining GH as a therapeutic target.

Purification, crystallization and preliminary X-ray analysis of human recombinant cytosolic serine hydroxymethyltransferase.

As an enzyme of the thymidylate synthase cycle, serine hydroxymethyltransferase (SHMT) has a key role in nucleotide biosynthesis. Elevated activities of SHMT have been correlated with the increased demand for nucleotide biosynthesis in tumors of human and rodent origin, making this enzyme a novel target for cancer chemotherapy. Here the purification and crystallization of recombinant human cytosolic SHMT are reported. Crystals belong to space group P6222 or P6422 with cell parameters a = b = 155.0, c = 235.5 A and diffract to at least 3.0 A resolution.

Characterization of human cellular gamma-glutamyl hydrolase.

A previously identified cDNA encoding a human gamma-glutamyl hydrolase was expressed in a baculovirus system. The expressed protein had molecular mass of 37 kDa. Treatment of the protein with PNGase F produced a protein of molecular mass of 30 kDa, indicating that the protein contained asparagine-linked glycosylation. Sequence analysis of the expressed protein indicated that a 24-amino-acid signal peptide had been removed. A polyclonal antibody to the expressed enzyme was used in Western blot analysis of partially purified lysates of HL-60 promyeloid leukemia cells and MCF-7 breast cancer cells. The HL-60 and MCF-7 enzymes appeared as two closely spaced bands with a molecular mass of 37 kDa. Treatment of the HL-60 enzyme with PNGase F produced a protein with a molecular mass of 30 kDa. The activities of the expressed enzyme and the enzyme from HL-60 cells were similar on methotrexate polyglutamates. Methotrexate-gamma-Glu is a poor substrate for the human enzyme relative to methotrexate gamma-Glu2-5. During hydrolysis of methotrexate-gamma-Glu4, all possible pterin-containing cleavage products (methotrexate and methotrexate-gamma-Glu1-3) appear. The results demonstrated that the human enzyme cleaves both the ultimate and penultimate gamma-linkages of methotrexate polyglutamates. Glutamate was released as either glutamic acid or gamma-Glu2. Longer chain species of gamma-Glun>2 were not observed. Inhibition by iodoacetic acid suggested that both the expressed enzyme and the HL-60 enzyme may contain a catalytically essential cysteine. These results indicate that the identified cDNA encodes the intracellular gamma-glutamyl hydrolase found in a variety of human tumor cells and that the baculovirus-expressed enzyme is a suitable model for further structural and enzymatic studies.

Characterisation of a human serine hydroxymethyltransferase pseudogene and its localisation to 1p32.3-33.

The conversion of serine and tetrahydrofolate to glycine and 5,10 methylene tetrahydrofolate by serine hydroxymethyltransferase (SHMT, EC 2.1.2.1) is the major route for the provision of one-carbon units for biosynthetic reactions. SHMT cDNAs have been cloned from both rabbit and man, and a human mitochondrial SHMT gene sequence has recently been reported. We have isolated phage clones containing human genomic sequences homologous to cytosolic SHMT and have found these to contain a processed pseudogene (SHMT-ps1) with a 90% identity to cloned SHMT cDNAs. SHMT-ps1 contains 2335 nt that are homologous to SHMT but it has an additional leader sequence of 203 nt of unknown origin. The complete SHMT-ps1 sequence of 2538 nt is bounded by two 16 nt direct repeats that are characteristic of retroelement insertion sites. Two phage clones containing SHMT-ps1 have been mapped by fluorescence in situ hybridisation to 1p32.3-33. In addition, an SHMT CDNA clone hybridized to the same region and to 17p11.2-12. The latter is consistent with a previous localisation of the gene for cytosolic SHMT.

Skeletal low-magnesium calcite in living scleractinian corals.

The skeletons of living specimens of the scleractinian coral Porites lobata have been found to contain up to 46 +/- 5 percent low-magnesium calcite even though free of gross detrital inclusions and boring or encrusting organisms. The calcite crystals occur in the interior of skeletal structures, have dimensions of 20 micrometers or less, and are surrounded by typical aragonite needles. Biogenic deposition seems to be the most likely source of the calcite, although the evidence does not rule out diagenesis of metastable.