Site-directed mutagenesis establishes cysteine-110 as essential for enzymeactivity in human gamma-glutamyl hydrolase

gamma-Glutamyl hydrolase (GH), which hydrolyses the gamma-glutamyl conjugat es of folic acid, is a key enzyme in the maintenance of cellular folylpolyg lutamate concentrations. The catalytic mechanism of GH is not known. Consis tent with earlier reports that GH is sulphydryl-sensitive, we found that re combinant human GH is inhibited by iodoacetic acid, suggesting that at leas t one cysteine is important for activity [Rhee, Lindau-Shepard, Chave, Gali van and Ryan (1998) Mol. Pharmacol. 53, 1040-1046]. Using site-directed mut agenesis, the cDNA for human GH was altered to encode four different protei ns 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 co nserved among the human, rat and mouse GH amino acid sequences. The wild-ty pe 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 o f the essential Cys residue in this enzyme and provide the beginning of a f ramework to determine the catalytic mechanism, important in defining GH as a therapeutic target.