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Titolo:
IDENTIFICATION OF AN ACTIVE ACIDIC RESIDUE IN THE CATALYTIC SITE OF BETA-HEXOSAMINIDASE
Autore:
TSE R; VAVOUGIOS G; HOU YM; MAHURAN DJ;
Indirizzi:
HOSP SICK CHILDREN,RES INST,555 UNIV AVE TORONTO ON M5G 1X8 CANADA HOSP SICK CHILDREN,RES INST TORONTO ON M5G 1X8 CANADA UNIV TORONTO,DEPT CLIN BIOCHEM TORONTO ON M5G 2C4 CANADA
Titolo Testata:
Biochemistry
fascicolo: 23, volume: 35, anno: 1996,
pagine: 7599 - 7607
SICI:
0006-2960(1996)35:23<7599:IOAAAR>2.0.ZU;2-J
Fonte:
ISI
Lingua:
ENG
Soggetto:
TAY-SACHS DISEASE; HUMAN PLACENTAL HEXOSAMINIDASE; N-ACETYLHEXOSAMINIDASE; SEQUENCE-ANALYSIS; ALPHA-SUBUNIT; GM2-GANGLIOSIDOSIS-B1 VARIANT; ENDOPLASMIC-RETICULUM; PARAMETER-ESTIMATION; EXTENSIVE HOMOLOGY; GM2 GANGLIOSIDOSIS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
56
Recensione:
Indirizzi per estratti:
Citazione:
R. Tse et al., "IDENTIFICATION OF AN ACTIVE ACIDIC RESIDUE IN THE CATALYTIC SITE OF BETA-HEXOSAMINIDASE", Biochemistry, 35(23), 1996, pp. 7599-7607

Abstract

Human beta-hexosaminidases A and B (EC 3.2.1.52) are dimeric lysosomal glycosidases composed of evolutionarily related alpha and/or beta subunits. Both isozymes hydrolyze terminal beta-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 alpha or beta subunits can result in G(M2) ganglioside storage and Tay-Sachs or Sandhoff disease, respectively. All glycosyl hydrolases are believed to have oneor 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 thatthis effect can be blocked by the inclusion of a strong competitive inhibitor in the reaction mix. We hypothesized that the catalytic acid reside(s) should be located in a region of overall homology and be invariant within the aligned deduced primary sequences of the human alphaand beta subunits, as well as hexosaminidases from other species, including bacteria. Such a region is encoded by exons 5-6 of the HEXA andHEXB genes. This region includes beta Arg(211) (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(alpha 258,beta 290), 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 beta cDNA, followed by cellular expression. Of these, only the beta Asp(196)Asn substitution decreased the k(cat) (350-910-fold) without any noticeable effect on the K-m. Mutagenesis of either beta Asp(240) or beta Asp(290) 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 beta Asp(196) is a catalytic acid residue in beta-hexosaminidase.

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Documento generato il 01/12/20 alle ore 12:12:06