Active site residues of human beta-glucuronidase - Evidence for Glu(540) as the nucleophile and Glu(451) as the acid-base residue

Human beta-glucuronidase (hGUSB) is a member of family 2 glycosylhydrolases that cleaves beta-D-glucuronic acid residues from the nonreducing termini of glycosaminoglycans. Amino acid sequence and structural homology of hGUSB and Escherichia coli beta-galactosidase active sites led us to propose tha t residues Glu(451), Glu(540), and Tyr(504), hGUSB are involved in catalysi s, Glu(451) being the acid-base residue and Glu(540) the nucleophile. To te st this hypothesis, we introduced mutations in these residues and determine d their effects on enzymes expressed in COS cells and GUSB-deficient fibrob lasts, The extremely low activity in cells expressing Glu(451), Glu(540), a nd Tyr(504) hGUSBs supported their roles in catalysis. For kinetic analysis , wild type and mutant enzymes were produced in baculovirus and purified to homogeneity by affinity chromatography. The k(cat)/K-m values (mM(-1).s(-1 )) of the E540A, E451A, and Y504A enzymes were 34,000-, 9100-, and 830-fold lower than that of wild type hGUSB, respectively. High concentrations of a zide stimulated the activity of the E451A mutant enzyme, supporting the rol e of Glu451 as the acid-base catalyst. We conclude that, like their homolog ues in E. coli beta-galactosidase, Glu(540) is the nucleophilic residue, Gl u(451) the acid-base catalyst, and Tyr(504) is, also important for catalysi s, although its role is unclear. All three residues are located in the acti ve site cavity previously determined by structural analysis of hGUSB.