Beta-galactosidase

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. Lactase is often confused as an alternative name for β-galactosidase, but it is actually simply a sub-class of β-galactosidase.

Properties and functions
β-galactosidase is an exoglycosidase which hydrolyzes the β-glycosidic bond formed between a galactose and its organic moiety. It may also cleave fucosides and arabinosides but with much lower efficiecy. It is an essential enzyme in the human body, deficiencies in the protein can result in galactosialidosis or Morquio B syndrome. In E. coli, the gene of β-galactosidase, the lacZ gene, is present as part of the inducible system lac operon which is activated in the presence of lactose when glucose level is low.

It is commonly used in molecular biology as reporter marker in various applications. Its exhibits a phenomenon called α-complementation which forms the basis for the blue/white screening of recombinant clones. This enzyme can be split in two peptides, LacZα and LacZΩ, neither of which is active by itself but when both are present together, spontaneously reassemble into a functional enzyme. This property is exploited in many cloning vectors where the presence of the lacZα gene in a plasmid can complement in trans another mutant gene encoding the LacZΩ in specific laboratory strains of E. coli. However, when DNA fragments are inserted in the vector, the production of LacZα is disrupted, the cells therefore show no β-galactosidase activity. The presence or absence of an active β-galactosidase may be detected by X-gal, which produces a characteristic blue dye when cleaved by β-galactosidase, thereby providing an easy means of distinguishing the presence or absence of cloned product in a plasmid.

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) which would be specifically expressed in senescence (The irreversible growth arrest of cells). Specific quantitative assays were even developed for its detection. However, it is now known that this is due to an overexpression and accumulation of the lysosomal endogenous beta-galactosidase, and its expression is not required for senescence. Nevertheless, it remains the most widely used biomarker for senescent and aging cells, because it is reliable and easy to detect.

Structure
The 1,024 amino acids of E. coli β-galactosidase were first sequenced in 1970, and its structure determined twenty-four years later in 1994. The protein is a 464-kDa homotetramer with 2,2,2-point symmetry Each unit of β-galactosidase consists of five domains; domain 1 is a jelly-roll type barrel, domain 2 and 4 are fibronectin type III-like barrels, domain 5 a β-sandwich, while the central domain 3 is a TIM-type barrel.

The third domain contains the active site. The active site is made up of elements from two subunits of the tetramer, and disassociation of the tetramer into dimers removes critical elements of the active site. The amino-terminal sequence of β-galactosidase, the α-peptide involved in α-complementation, participates in a subunit interface. Its residues 22-31 help to stabilize a four-helix bundle which forms the major part of that interface, and residue 13 and 15 also contributing to the activating interface. These structural features provide a rationale for the phenomenon of α-complementation, where the deletion of the amino-terminal segment results in the formation of an inactive dimer.

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. However, it is now known that Glu-461 is an acid catalyst. Instead, Glu-537 is the actual nucleophile, binding to a galactosyl intermediate.

In humans, the nucleophile of the hydrolysis reaction is Glu-268.

Uses
β-galactosidase assay is used frequently in genetics, molecular biology, and other life sciences. An active enzyme may be detected using X-gal, which forms an intense blue product after cleavage by β-galactosidase, and is easy to identify and quantify. It is used for example in blue white screen. Its production may be induced by a non-hydrolyzable analog of allolactose, IPTG, which binds and releases the lac repressor from the lac operator, thereby allowing the initiation of transcription to proceed.

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 assays, despite its limitations.