Molecular Modeling and Site-directed Mutagenesis Define the Catalytic Motif in Human γ-Glutamyl Hydrolase

Karen J. Chave, Ivan E. Auger, John Galivan, Thomas J. Ryan
2000 Journal of Biological Chemistry  
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
more » ... 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.
doi:10.1074/jbc.m007908200 pmid:11005824 fatcat:u32uqe3lwncenbgzr2t3y3454a