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

Biochemistry
Y HouD J Mahuran

Abstract

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 with other evolutionarily related glycosidases into "Family 20". The three-dimensional structure of only one Family 20 member has been elucidated, a bacterial chitobiase. This enzyme shares primary structure homology with both the human subunits only in its active-site region, and even in this restricted area, the level of identity is only 26%. Thus, the validity of the molecular model for the active site of the human enzyme based on chitobiase must be determined experimentally. In this report, we analyze highly purified mutant forms of human hexosaminidase B that have had conservative substitutions made at Glu and Asp residues predicted by the chitobiase model to be part 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/base catalytic residue. Similarly, mutation of beta Asp(354) to Asn reduced k(cat) 2000-fold while leaving K(m) essentially unaltered, consistent with assignment of this residue as the residue that i...Continue Reading

References

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Citations

Mar 14, 2003·Journal of Molecular Biology·Pirkko HeikinheimoEdward Hough
May 24, 2008·Glycobiology·Brian P Rempel, Stephen G Withers
Mar 13, 2013·The Journal of Biological Chemistry·Tasuku ItoShinya Fushinobu
Mar 12, 2016·Molecular Therapy. Methods & Clinical Development·Michael B TropakDon Mahuran
Oct 16, 2004·Biochemical and Biophysical Research Communications·Maryam ZarghooniDon Mahuran
May 6, 2016·Biochemistry·Matthew G AlteenTracey M Gloster
Apr 15, 2004·Experimental Neurology·Douglas R MartinHenry J Baker
Mar 19, 2004·The Journal of Biological Chemistry·Mattias Collin, Vincent A Fischetti
Dec 2, 2008·The Biochemical Journal·Martin GutterniggIain B H Wilson
Jan 29, 2020·International Journal of Biological Macromolecules·Ashapogu VenugopalMusti J Swamy
Jan 10, 2020·ACS Chemical Biology·Alexander EddendenMark Nitz

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