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.
Chave
K J
KJ
Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509, USA.
Galivan
J
J
Ryan
T J
TJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D017124:Conserved Sequence; D003545:Cysteine; D017931:DNA Primers; D006801:Humans; D019807:Iodoacetic Acid / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D011623:gamma-Glutamyl Hydrolase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D011623:gamma-Glutamyl Hydrolase; D003545:Cysteine; D019807:Iodoacetic Acid