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 anti folylpoly-gamma -glutamate metabolism, which functions by catalyzing the cl eavage of the gamma -glutamyl chain of substrates. We previously reported t hat 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 gluta mine 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. Th is model predicts that Cys-110 of hGH is the active site nucleophile and fo rms a catalytic triad with residues His-220 and Glu-222. The hGH mutants C1 10A, H220A, and E222A were prepared. Consistent with the model, mutants C11 0A and H220A were inactive. However, the V-max of the E222A hGH mutant was reduced only g-fold relative to the wild-type enzyme. The model also predic ted 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 determ ine the catalytic mechanism begin to define the three dimensional interacti ons of hGH with poly-gamma -glutamate substrates.