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.
Chave
K J
KJ
Division of Molecular Medicine, Wadsworth Center, New York State Department of of Health, Albany, New York 12201-0509, USA.
Auger
I E
IE
Galivan
J
J
Ryan
T J
TJ
D020816:Amino Acid Motifs; D001483:Base Sequence; D020134:Catalytic Domain; D017931:DNA Primers; D005492:Folic Acid / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000627:therapeutic use; D006706:Homeostasis; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011623:gamma-Glutamyl Hydrolase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D005493:Folic Acid Antagonists; D005492:Folic Acid; D011623:gamma-Glutamyl Hydrolase