Three-dimensional structure of human γ-glutamyl hydrolase: A class I glutamine amidotransferase adapted for a complex substrate

γ-Glutamyl hydrolase catalyzes the cleavage of the γ-glutamyl chain of folylpoly-γ-glutamyl substrates and is a central enzyme in folyl and antifolyl poly-γ-glutamate metabolism. The crystal structure of human γ-glutamyl hydrolase, determined at 1.6-Å resolution, reveals that the protein is a homodimer. The overall structure of human γ-glutamyl hydrolase contains 11 α-helices and 14 β-strands, with a fold in which a central eight-stranded β-sheet is sandwiched by three and five α-helices on each side. The topology is very similar to that of the class I glutamine amidotransferase domains, with the only major differences consisting of extensions in four loops and at the C terminus. These insertions are important for defining the substrate binding cleft and/or the dimer interface. Two sequence motifs are found in common between human γ-glutamyl hydrolase and the class I glutamine amidotransferase family and include the catalytically essential residues, Cys-110 and His-220. These residues are located in the center of a large L-shaped cleft that is closed at one end and open at the other. Several conserved residues, including Glu-114, His-171, Gln-218, and Lys-223, may be important for substrate binding. Modeling of a methotrexate thioester intermediate, based on the corresponding complex of the glutamate thioester intermediate of Escherichia coli carbamoyl-phosphate synthetase, indicates that the substrate binds in an orientation with the pteroyl group toward the open end of the cleft.