Characterization of the Glu and Asp residues in the active site of human beta-hexosaminidase B

Human beta -hexosaminidase A (alpha beta) and B (beta beta) are composed of subunits (alpha and beta) that are 60% identical and have been grouped wit h other evolutionarily related glycosidases into,"Family 20". The three-dim ensional structure of only one Family 20 member has been elucidated, a bact erial chitobiase. This enzyme shares primary structure homology with both t he human subunits only in its active-site region, and even in this restrict ed area, the level of identity is only 26%. Thus, the validity of the molec ular model for the active site of the human enzyme based on chitobiase must be determined experimentally. In this report, we analyze highly purified m utant forms of human hexosaminidase B that have had conservative substituti ons made at Glu and Asp residues predicted by the chitobiase model to be pa rt of its active site. Mutation of beta Glu(355) to Gln reduces k(cat) 5000 -fold with only a small effect on K-m, while also shifting the pH optimum. These effects are consistent with assignment of this residue as the acid/ba se catalytic residue. Similarly, mutation of beta Asp(354) to Asn reduced k (cat) 2000-fold while leaving K-m essentially unaltered, consistent with as signment of this residue as the residue that interacts with the substrate a cetamide group to promote its attack on the anomeric center. These data in conjunction with the mutagenesis studies of Asp(241) and Glu(491) indicate that the molecular model is substantially accurate in its identification of catalytically important residues.