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β-galactosidase, also called beta-gal or β-gal, is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins.[1] Alternate or nicknames are "beta-gal" or "β-gal". Lactase is often confused as an alternative name for β-galactosidase, but it is actually simply a sub-class of β-galactosidase.
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Structure
The 1,021 amino acids of E. coli β-galactosidase were first sequenced in 1970.[2] Four such chains comprise the protein, which was discovered to be a 464-kDa tetramer with 222-point symmetry twenty-four years later. Each unit of β-galactosidase consists of five domains, the third of which is an active site.[3] This enzyme can be split in two peptides, LacZα and LacZΩ, none of which is active by itself but both spontaneously reassemble into a functional enzyme. This characteristic is used in many cloning vectors to achieve α-complementation in specific laboratory strains of E. coli, where the small LacZα peptide is encoded by the plasmid while the large LacZΩ is encoded in trans by the bacterial chromosome. When DNA fragments are inserted in the vector and production of LacZα is disrupted, the cells exhibit no β-galactosidase activity: this allows the blue/white screening of recombinant clones.
In 1995, Dimri et al. proposed a new isoform for beta-galactosidase with optimum activity at pH 6.0 (Senescence Associated beta-gal or SA-beta-gal)[4] which would be specifically expressed in senescence (The irreversible growth arrest of cells). Even specific quantitative assays were developed for its detection.[5][6][7] Today it is known that this corresponds to an accumulation of the lysosomal endogenous beta-galactosidase.[8] and its expression is not required for senescence. However, it remains as the most widely used biomarker for senescent and aging cells, because it is reliable and easy to detect.
Reaction
The active site of β-galactosidase catalyzes the hydrolysis of its disaccharide substrate via "shallow" and "deep" binding. Monovalent potassium ions (K+) as well as divalent magnesium ions (Mg2+) are required for the enzyme's optimal activity. The beta-linkage of the substrate is cleaved by a terminal carboxyl group on the side chain of a glutamic acid.
In E. coli, Glu-461 was thought to be the nucleophile in the substitution reaction.[9] However, it is now known that Glu-461 is an acid catalyst. Instead, Glu-537 is the actual nucleophile,[10] binding to a galactosyl intermediate.
In humans, the nucleophile of the hydrolysis reaction is Glu-268.[11]
Biology
β-galactosidase is an essential enzyme in the human body. Deficiencies in the protein can result in galactosialidosis or Morquio B syndrome.
In E. coli, β-galactosidase is produced by activation of the lac operon, as the lacZ gene.
Other Uses
β-galactosidase assay is used frequently in genetics, molecular biology (see X-gal), and other life sciences: [12]
IPTG induces production of β-galactosidase by binding and inhibiting the lac repressor.
Since it is highly expressed and accumuled in lysosomes in senescent cells, it is used as a senescence biomarker both in vivo and in vitro in qualitative and quantitative [5] assays, despite its limitations
References
- ^ Dorland's Illustrated Medical Dictionary. http://www.mercksource.com/pp/us/cns/cns_hl_dorlands.jspzQzpgzEzzSzppdocszSzuszSzcommonzSzdorlandszSzdorlandzSzdmd_g_01zPzhtm#12382991. Retrieved 2006-10-22.
- ^ Fowler AV, Zabin I (October 1970). "The amino acid sequence of beta galactosidase. I. Isolation and composition of tryptic peptides". J. Biol. Chem. 245 (19): 5032–41. PMID 4918568. http://www.jbc.org/cgi/reprint/245/19/5032.
- ^ Matthews BW (June 2005). "The structure of E. coli beta-galactosidase". C. R. Biol. 328 (6): 549–56. doi:. PMID 15950161.
- ^ Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O (September 1995). "A biomarker that identifies senescent human cells in culture and in aging skin in vivo". Proc. Natl. Acad. Sci. U.S.A. 92 (20): 9363–7. PMID 7568133.
- ^ a b Bassaneze V, Miyakawa AA, Krieger JE (January 2008). "A quantitative chemiluminescent method for studying replicative and stress-induced premature senescence in cell cultures". Anal. Biochem. 372 (2): 198–203. doi:. PMID 17920029.
- ^ Gary RK, Kindell SM (August 2005). "Quantitative assay of senescence-associated beta-galactosidase activity in mammalian cell extracts". Anal. Biochem. 343 (2): 329–34. doi:. PMID 16004951.
- ^ Itahana K, Campisi J, Dimri GP (2007). "Methods to detect biomarkers of cellular senescence: the senescence-associated beta-galactosidase assay". Methods Mol. Biol. 371: 21–31. PMID 17634571.
- ^ Lee BY, Han JA, Im JS, Morrone A, Johung K, Goodwin EC, Kleijer WJ, DiMaio D, Hwang ES (April 2006). "Senescence-associated beta-galactosidase is lysosomal beta-galactosidase". Aging Cell 5 (2): 187–95. doi:. PMID 16626397.
- ^ Gebler JC, Aebersold R, Withers SG (June 1992). "Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) beta-galactosidase from Escherichia coli". J. Biol. Chem. 267 (16): 11126–30. PMID 1350782. http://www.jbc.org/content/267/16/11126.long.
- ^ Yuan J, Martinez-Bilbao M, Huber RE (April 1994). "Substitutions for Glu-537 of beta-galactosidase from Escherichia coli cause large decreases in catalytic activity". Biochem. J. 299 ( Pt 2): 527–31. PMID 7909660.
- ^ McCarter JD, Burgoyne DL, Miao S, Zhang S, Callahan JW, Withers SG (January 1997). "Identification of Glu-268 as the catalytic nucleophile of human lysosomal beta-galactosidase precursor by mass spectrometry". J. Biol. Chem. 272 (1): 396–400. PMID 8995274.
- ^ Beta-Galactosidase Assay (A better Miller) - OpenWetWare
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