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Human ␥-glutamyl hydrolase (hGH) is a central enzyme in folyl and antifolylpoly-␥-glutamate metabolism, which functions by catalyzing the cleavage of the ␥-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-␥-glutamate substrates. ␥-Glutamyl hydrolase (GH)1 (EC 3.4.19.9), which hydrolyses the ␥-glutamyl conjugates of folic acid and antifolates, is a key enzyme in the metabolism of folic acid and the pharmacology of antifolates. Folate is required as a cofactor by several enzymes in the de novo biosynthesis of DNA precursors and several amino acids. Antifolates (for example, methotrexate (MTX)) have been the traditional treatment for many cancers. When the cell takes up folates or antifolates, they are poly-␥-glutamylated by the enzyme folylpolyglutamate synthetase (EC 6.3.2.17) (1). These polyglutamates are retained intracellularly and are generally better substrates or inhibitors of the target enzymes (2-4). GH alters these properties by catalyzing the removal of the polyglutamate chain (1, 5, 6).Although GH is known to cleave folylpolyglutamates, ultimately yielding folylmonoglutamate and glutamate, its role in the intracellular folate-dependent pathways remains unclear. Similarly, the role of GH in folate deficiencies is ambiguous. Increased GH activity (7,8) or decreased folylpolyglutamate synthetase activity (9 -12) can produce resistance to MTX in vitro. Recently, the ratio of folylpolyglutamate synthetase and GH activities has been demonstrated to be an indicator of MTX polyglutamylation in acute lymphocytic leukemia (13), and inherent resistance to MTX in acute myelogenous leukemia (14) may be due to an increased GH activity. However, clarification of the role of GH in these outcomes is unavailable. Therefore, definition of the active site residues and mechanism of human GH (hGH) will be necessary steps in developing specific inhibitors to assess its cellular function.Despite the important role of GH in folate function and antifolate therapeutics, no...