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Article

Identification of an Active Acidic Residue in the Catalytic Site of β-Hexosaminidase^†

Roderick Tse,^‡^§ George Vavougios,^‡^§ Yongmin Hou,^‡^§ and Don J. Mahuran*^‡^§

Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, and Department of Clinical Biochemistry, University of Toronto, Toronto, Ontario M5G 2C4, Canada

Biochemistry, 1996, 35 (23), pp 7599–7607

DOI: 10.1021/bi960246+

Publication Date (Web): June 11, 1996

†

This work was funded through a grant to D.M. from the Medical Research Council of Canada.

‡

Department of Clinical Biochemistry, University of Toronto.

Research Institute, Hospital for Sick Children.

To whom correspondence should be addressed at Research Institute, The Hospital For Sick Children, 555 University Avenue, Toronto, Ontario, Canada, M5G 1X8. Telephone: 416-813-6161. Fax: 416-813-5086.

Abstract

Human β-hexosaminidases A and B (EC 3.2.1.52) are dimeric lysosomal glycosidases composed of evolutionarily related α and/or β subunits. Both isozymes hydrolyze terminal β-linked GalNAc or GlcNAc residues from numerous artificial and natural substrates; however, in vivo G_M2 ganglioside is a substrate for only the heterodimeric A isozyme. Thus, mutations in either gene encoding its α or β subunits can result in G_M2 ganglioside storage and Tay-Sachs or Sandhoff disease, respectively. All glycosyl hydrolases are believed to have one or more acidic residues in their catalytic site. We demonstrate that incubation of hexosaminidase with a chemical modifier specific for carboxyl side chains produces a time-dependent loss of activity, and that this effect can be blocked by the inclusion of a strong competitive inhibitor in the reaction mix. We hypothesized that the catalytic acid residue(s) should be located in a region of overall homology and be invariant within the aligned deduced primary sequences of the human α and β subunits, as well as hexosaminidases from other species, including bacteria. Such a region is encoded by exons 5−6 of the HEXA and HEXB genes. This region includes βArg₂₁₁ (invariant in 15 sequences), which we have previously shown to be an active residue. This region also contains two invariant and one conserved acidic residues. A fourth acidic residue, Asp^α^258,^β²⁹⁰, in exon 7 was also investigated because of its association with the B1 variant of Tay-Sachs disease. Conservative substitutions were made at each candidate residue by in vitro mutagenesis of a βcDNA, followed by cellular expression. Of these, only the βAsp¹⁹⁶Asn substitution decreased the k_cat (350−910-fold) without any noticeable effect on the K_m. Mutagenesis of either βAsp²⁴⁰ or βAsp²⁹⁰ to Asn decreased k_cat by 10- or 1.4-fold but also raised the K_m of the enzyme 11- or 3- fold, respectively. The above results strongly suggest that βAsp¹⁹⁶ is a catalytic acid residue in β-hexosaminidase.