β-Galactosidase

β-Galactosidase (EC 3.2.1.23, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase) is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. (This enzyme digests many β-Galactosides, not just lactose. It is sometimes loosely referred to as lactase but that name is generally reserved for mammalian digestive enzymes that breaks down lactose specifically.)

β-Galactosides include carbohydrates containing galactose where the glycosidic bond lies above the galactose molecule. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins.<ref name=dorland>Template:Cite book</ref>

Function

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 at a much lower rate. 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 lacZ gene is the structural gene for β-galactosidase; which is present as part of the inducible system lac operon which is activated in the presence of lactose when glucose level is low. β-Galactosidase synthesis stops when glucose levels are sufficient.<ref>Template:Cite book</ref>

β-Galactosidase has many homologues based on similar sequences. A few are evolved β-galactosidase (EBG), β-glucosidase, 6-phospho-β-galactosidase, β-mannosidase, and lactase-phlorizin hydrolase. Although they may be structurally similar, they all have different functions.<ref name="IPR017736">Template:Cite web</ref> Beta-gal is inhibited by L-ribose and by competitive inhibitors 2-phenylethyl 1-thio-β-D-galactopyranoside (PETG), D-galactonolactone, isopropyl thio-β-D-galactoside (IPTG), and galactose.<ref name="Juers_2003">Template:Cite journal</ref>

β-Galactosidase is important for organisms as it is a key provider in the production of energy and a source of carbons through the break down of lactose to galactose and glucose. It is also important for lactose-intolerant people as it is responsible for making lactose-free milk and other dairy products. Many adult humans lack the lactase enzyme, which has the same function as β-galactosidase, so they are not able to properly digest dairy products. β-Galactose is used in such dairy products as yogurt, sour cream, and some cheeses which are treated with the enzyme to break down any lactose before human consumption. In recent years, β-galactosidase has been researched as a potential treatment for lactose intolerance through gene replacement therapy where it could be placed into the human DNA so individuals can break down lactose on their own.<ref>Template:Cite journal</ref><ref>Template:Cite journal</ref>

Structure

The 1,023 amino acids of E. coli β-galactosidase were sequenced in 1983,<ref>Template:Cite journal</ref> and its structure determined eleven years later in 1994. The protein is a 464-kDa homotetramer with 2,2,2-point symmetry.<ref name="Jacobson_1994">Template:Cite journal</ref> Each unit of β-galactosidase consists of five domains; domain 1 is a jelly-roll type β-barrel, domain 2 and 4 are fibronectin type III-like barrels, domain 5 a novel β-sandwich, while the central domain 3 is a distorted TIM-type barrel, lacking the fifth helix with a distortion in the sixth strand.<ref name="Jacobson_1994" />

The third domain contains the active site.<ref name="pmid15950161">Template:Cite journal</ref> 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.Template:Citation needed 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

File:Beta-galactosidase.png

β-galactosidase reaction

β-Galactosidase can catalyze three different reactions in organisms. In one, it can go through a process called transgalactosylation to make allolactose, creating a positive feedback loop for the production of β-galactose. Allolactose can also be cleaved to form monosaccharides. It can also hydrolyze lactose into galactose and glucose which will proceed into glycolysis.<ref name="IPR017736" /> The active site of β-galactosidase catalyzes the hydrolysis of its disaccharide substrate via "shallow" (nonproductive site) and "deep" (productive site) binding. Galactosides such as PETG and IPTG will bind in the shallow site when the enzyme is in "open" conformation while transition state analogs such as L-ribose and D-galactonolactone will bind in the deep site when the conformation is "closed".<ref name="Juers_2003" />

The enzymatic reaction consists of two chemical steps, galactosylation and degalactosylation. Galactosylation is the first chemical step in the reaction where Glu461 donates a proton to a glycosidic oxygen, resulting in galactose covalently bonding with Glu537. In the second step, degalactosylation, the covalent bond is broken when Glu461 accepts a proton, replacing the galactose with water. Two transition states occur in the deep site of the enzyme during the reaction, once after each step. When water participates in the reaction, galactose is formed, otherwise, when D-glucose acts as the acceptor in the second step, transgalactosylation occurs .<ref name="Juers_2003" /> It has been kinetically measured that single tetramers of the protein catalyze reactions at a rate of 38,500 ± 900 reactions per minute.<ref name="Juers_2012">Template:Cite journal</ref> Monovalent potassium ions (K⁺) as well as divalent magnesium ions (Mg²⁺) are required for the enzyme's optimal activity. The β-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.<ref name="pmid1350782">Template:Cite journal</ref> However, it is now known that Glu-461 is an acid catalyst. Instead, Glu-537 is the actual nucleophile,<ref name="pmid7909660">Template:Cite journal</ref> binding to a galactosyl intermediate. In humans, the nucleophile of the hydrolysis reaction is Glu-268.<ref name="pmid8995274">Template:Cite journal</ref> Gly794 is important for β-galactosidase activity. It is responsible for putting the enzyme in a "closed", ligand bounded, conformation or "open" conformation, acting like a "hinge" for the active site loop. The different conformations ensure that only preferential binding occurs in the active site. In the presence of a slow substrate, Gly794 activity increased as well as an increase in galactosylation and decrease in degalactosylation.<ref name="Juers_2003" />

Applications

The β-galactosidase assay is used frequently in genetics, molecular biology, and other life sciences.<ref>Template:Cite book</ref> An active enzyme may be detected using artificial chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, X-gal. β-galactosidase will cleave the glycosidic bond in X-gal and form galactose and 5-bromo-4-chloro-3-hydroxyindole which dimerizes and oxidizes to 5,5'-dibromo-4,4'-dichloro-indigo, an intense blue product that is easy to identify and quantify.<ref name="pmid16004951"/> It is used for example in blue white screen.<ref>β-Galactosidase Assay (A better Miller) - OpenWetWare</ref> 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.

It is commonly used in molecular biology as a reporter marker to monitor gene expression. It also 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 studies of leukaemia chromosomal translocations, Dobson and colleagues used a fusion protein of LacZ in mice,<ref>Template:Cite journal</ref> exploiting β-galactosidase's tendency to oligomerise to suggest a potential role for oligomericity in MLL fusion protein function.<ref>Template:Cite journal</ref>

A recent study conducted in 2020–2021 determined that Beta-Galactosidase activity correlates with senescence of the cells. Senescence of the cells can be interpreted as cells that do not divide, but cells that do not die. Beta-Galactosidase activity can be overexpressed, and this can lead to various diseases afflicting a wide range of body systems. These systems include the cardiovascular system, skeletal system, and many more. Detecting senescence cells can be achieved by measuring the lysosomal Beta-Galactosidase activity.<ref>Template:Cite journal</ref>

A new isoform for beta-galactosidase with optimum activity at pH 6.0 (Senescence Associated beta-gal or SA-beta-gal) <ref name="pmid7568133">Template:Cite journal</ref> which is specifically expressed in senescence (the irreversible growth arrest of cells). Specific quantitative assays were even developed for its detection.<ref name="VB">Template:Cite journal</ref><ref name="pmid16004951">Template:Cite journal</ref><ref name="pmid17634571">Template:Cite book</ref> However, it is now known that this is due to an overexpression and accumulation of the lysosomal endogenous beta-galactosidase,<ref name="pmid16626397">Template:Cite journal</ref> 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.

Evolution

Some species of bacteria, including E. coli, have additional β-galactosidase genes. A second gene, called evolved β-galactosidase (ebgA) gene was discovered when strains with the lacZ gene deleted (but still containing the gene for galactoside permease, lacY), were plated on medium containing lactose (or other 3-galactosides) as sole carbon source. After a time, certain colonies began to grow. However, the EbgA protein is an ineffective lactase and does not allow growth on lactose. Two classes of single point mutations dramatically improve the activity of ebg enzyme toward lactose.<ref name="Hall1977">Template:Cite journal</ref><ref name="Hall1981">Template:Cite journal</ref> and, as a result, the mutant enzyme is able to replace the lacZ β-galactosidase.<ref name="Hall1976">Template:Cite journal</ref> EbgA and LacZ are 50% identical on the DNA level and 33% identical on the amino acid level.<ref name="Hall1985">Template:Cite journal</ref> The active ebg enzyme is an aggregate of ebgA -gene and ebgC-gene products in a 1:1 ratio with the active form of ebg enzymes being an α4 β4 hetero-octamer.<ref name="Elliott1992">Template:Cite journal</ref>

Species distribution

Much of the work done on β-galactosidase is derived from E. coli. However the enzyme can be found in many plants (especially fruits), mammals, yeast, bacteria, and fungi.<ref>Template:Cite journal</ref> β-galactosidase genes can differ in the length of their coding sequence and the length of proteins formed by amino acids.<ref name = "Guo_2018">Template:Cite journal</ref> This separates the β-galactosidases into four families: GHF-1, GHF-2, GHF-35, and GHF- 42.<ref name="Rojas_2004">Template:Cite journal</ref> E. Coli belongs to GHF-2, all plants belong to GHF-35, and Thermus thermophilus belongs to GHF-42.<ref name="Rojas_2004" /><ref name = "Guo_2018" /> Various fruits can express multiple β-galactosidase genes. There are at least seven β-galactosidase genes expressed in tomato fruit development, that have amino acid similarity between 33% and 79%.<ref>Template:Cite journal</ref> A study targeted at identifying fruit softening of peaches found 17 different gene expressions of β-galactosidases.<ref name="Guo_2018" /> The only other known crystal structure of β-galactosidase is from Thermus thermophilus. <ref name="Rojas_2004" />

External links

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