Beta-galactosidase
Encyclopedia
β-galactosidase, also called beta-gal or β-gal, is a hydrolase
enzyme
that catalyzes the hydrolysis
of β-galactosides into monosaccharide
s. Substrate
s of different β-galactosidases include ganglioside
GM1, lactosylceramides, lactose
, and various glycoprotein
s. Lactase
is often confused as an alternative name for β-galactosidase, but it is actually simply a sub-class of β-galactosidase.
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 efficiency. 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 a reporter marker
in various applications. It 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 vector
s 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.
s 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.
substrate via "shallow" and "deep" binding. Monovalent potassium
ion
s (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 human
s, the nucleophile
of the hydrolysis
reaction is Glu-268.
, 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 accumulated 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.
Hydrolase
In biochemistry, a hydrolase is an enzyme that catalyzes the hydrolysis of a chemical bond. For example, an enzyme that catalyzed the following reaction is a hydrolase:-Nomenclature:...
enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
that catalyzes the hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
of β-galactosides into monosaccharide
Monosaccharide
Monosaccharides are the most basic units of biologically important carbohydrates. They are the simplest form of sugar and are usually colorless, water-soluble, crystalline solids. Some monosaccharides have a sweet taste. Examples of monosaccharides include glucose , fructose , galactose, xylose...
s. Substrate
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
s of different β-galactosidases include ganglioside
Ganglioside
Ganglioside is a molecule composed of a glycosphingolipid with one or more sialic acids linked on the sugar chain. The 60+ known gangliosides differ mainly in the position and number of NANA residues.It is a component of the cell plasma membrane that modulates cell signal transduction events...
GM1, lactosylceramides, lactose
Lactose
Lactose is a disaccharide sugar that is found most notably in milk and is formed from galactose and glucose. Lactose makes up around 2~8% of milk , although the amount varies among species and individuals. It is extracted from sweet or sour whey. The name comes from or , the Latin word for milk,...
, and various glycoprotein
Glycoprotein
Glycoproteins are proteins that contain oligosaccharide chains covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation. In proteins that have segments extending...
s. Lactase
Lactase
Lactase , a part of the β-galactosidase family of enzymes, is a glycoside hydrolase involved in the hydrolysis of the disaccharide lactose into constituent galactose and glucose monomers...
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 exoglycosidaseExoglycosidase
A exoglycosidase is a glycoside hydrolase enzyme which breaks the glycosidic bonds at the terminal residue....
which hydrolyzes the β-glycosidic bond
Glycosidic bond
In chemistry, a glycosidic bond is a type of covalent bond that joins a carbohydrate molecule to another group, which may or may not be another carbohydrate....
formed between a galactose
Galactose
Galactose , sometimes abbreviated Gal, is a type of sugar that is less sweet than glucose. It is a C-4 epimer of glucose....
and its organic moiety. It may also cleave fucosides
Fucose
Fucose is a hexose deoxy sugar with the chemical formula C6H12O5. It is found on N-linked glycans on the mammalian, insect and plant cell surface, and is the fundamental sub-unit of the fucoidan polysaccharide...
and arabinosides
Arabinose
Arabinose is an aldopentose – a monosaccharide containing five carbon atoms, and including an aldehyde functional group.For biosynthetic reasons, most saccharides are almost always more abundant in nature as the "D"-form, or structurally analogous to D-glyceraldehyde.For sugars, the D/L...
but with much lower efficiency. It is an essential enzyme in the human body, deficiencies in the protein can result in galactosialidosis
Galactosialidosis
Galactosialidosis is a lysosomal storage disease.It is associated with cathepsin A.-External links:*...
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
Lac operon
The lac operon is an operon required for the transport and metabolism of lactose in Escherichia coli and some other enteric bacteria. It consists of three adjacent structural genes, lacZ, lacY and lacA. The lac operon is regulated by several factors including the availability of glucose and of...
which is activated in the presence of lactose when glucose level is low.
It is commonly used in molecular biology as a reporter marker
Reporter gene
In molecular biology, a reporter gene is a gene that researchers attach to a regulatory sequence of another gene of interest in cell culture, animals or plants. Certain genes are chosen as reporters because the characteristics they confer on organisms expressing them are easily identified and...
in various applications. It exhibits a phenomenon called α-complementation which forms the basis for the blue/white screening
Blue white screen
The blue-white screen is a screening technique that allows for the detection of successful ligations in vector-based gene cloning. DNA of interest is ligated into a vector. The vector is then transformed into competent cell . The competent cells are grown in the presence of X-gal...
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 vector
Cloning vector
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with a restriction enzyme that creates the same overhang, then ligating the fragments...
s where the presence of the lacZα gene in a plasmid can complement in trans
Trans-acting
In the field of molecular biology, trans-acting , in general, means "acting from a different molecule"...
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
X-gal
X-gal is an organic compound consisting of galactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and collaborators in Detroit, MI, in 1964. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl...
, 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
SA-beta-gal
SA-β-gal is a hypothetical hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides only in senescent cells....
) which would be specifically expressed in senescence
Senescence
Senescence or biological aging is the change in the biology of an organism as it ages after its maturity. Such changes range from those affecting its cells and their function to those affecting the whole organism...
(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 acidAmino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s of E. coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
β-galactosidase were first sequenced in 1970, and its structure determined twenty-four years later in 1994. The protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
is a 464-kDa
Atomic mass unit
The unified atomic mass unit or dalton is a unit that is used for indicating mass on an atomic or molecular scale. It is defined as one twelfth of the rest mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state, and has a value of...
homotetramer
Homotetramer
A homotetramer is a protein complex made up of four identical subunits which are associated but not covalently bound. A heterotetramer is a 4-subunit complex where one or more subunits differ....
with 2,2,2-point symmetry
Symmetry
Symmetry generally conveys two primary meanings. The first is an imprecise sense of harmonious or aesthetically pleasing proportionality and balance; such that it reflects beauty or perfection...
Each unit of β-galactosidase consists of five domains
Protein domain
A protein domain is a part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded. Many proteins consist of several structural...
; domain 1 is a jelly-roll type barrel
Beta barrel
A beta barrel is a large beta-sheet that twists and coils to form a closed structure in which the first strand is hydrogen bonded to the last.Beta-strands in beta-barrels are typically arranged in an antiparallel fashion...
, domain 2 and 4 are fibronectin type III
Fibronectin type III domain
The Fibronectin type III domain is an evolutionary conserved protein domain that is widely found in animal proteins. The fibronectin protein in which this domain was first identified contains 16 copies of this domain. The domain is about 100 amino acids long and possesses a beta sandwich...
-like barrels, domain 5 a β-sandwich, while the central domain 3 is a TIM-type barrel
TIM barrel
The TIM barrel is a conserved protein fold consisting of eight α-helices and eight parallel β-strands that alternate along the peptide backbone. The structure is named after triosephosphate isomerase, a conserved glycolytic enzyme. TIM barrels are quite common among the conserved protein folds...
.
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 disaccharideDisaccharide
A disaccharide or biose is the carbohydrate formed when two monosaccharides undergo a condensation reaction which involves the elimination of a small molecule, such as water, from the functional groups only. Like monosaccharides, disaccharides form an aqueous solution when dissolved in water...
substrate via "shallow" and "deep" binding. Monovalent potassium
Potassium
Potassium is the chemical element with the symbol K and atomic number 19. Elemental potassium is a soft silvery-white alkali metal that oxidizes rapidly in air and is very reactive with water, generating sufficient heat to ignite the hydrogen emitted in the reaction.Potassium and sodium are...
ion
Ion
An ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...
s (K+) as well as divalent magnesium
Magnesium
Magnesium is a chemical element with the symbol Mg, atomic number 12, and common oxidation number +2. It is an alkaline earth metal and the eighth most abundant element in the Earth's crust and ninth in the known universe as a whole...
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
Side chain
In organic chemistry and biochemistry, a side chain is a chemical group that is attached to a core part of the molecule called "main chain" or backbone. The placeholder R is often used as a generic placeholder for alkyl group side chains in chemical structure diagrams. To indicate other non-carbon...
of a glutamic acid
Glutamic acid
Glutamic acid is one of the 20 proteinogenic amino acids, and its codons are GAA and GAG. It is a non-essential amino acid. The carboxylate anions and salts of glutamic acid are known as glutamates...
.
In E. coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
, Glu-461 was thought to be the nucleophile
Nucleophile
A nucleophile is a species that donates an electron-pair to an electrophile to form a chemical bond in a reaction. All molecules or ions with a free pair of electrons can act as nucleophiles. Because nucleophiles donate electrons, they are by definition Lewis bases.Nucleophilic describes the...
in the substitution reaction. However, it is now known that Glu-461 is an acid
Acid
An acid is a substance which reacts with a base. Commonly, acids can be identified as tasting sour, reacting with metals such as calcium, and bases like sodium carbonate. Aqueous acids have a pH of less than 7, where an acid of lower pH is typically stronger, and turn blue litmus paper red...
catalyst. Instead, Glu-537 is the actual nucleophile, binding to a galactosyl intermediate.
In human
Human
Humans are the only living species in the Homo genus...
s, the nucleophile
Nucleophile
A nucleophile is a species that donates an electron-pair to an electrophile to form a chemical bond in a reaction. All molecules or ions with a free pair of electrons can act as nucleophiles. Because nucleophiles donate electrons, they are by definition Lewis bases.Nucleophilic describes the...
of the hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
reaction is Glu-268.
Uses
The β-galactosidase assay is used frequently in geneticsGenetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
, molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
, and other life sciences. An active enzyme may be detected using X-gal
X-gal
X-gal is an organic compound consisting of galactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and collaborators in Detroit, MI, in 1964. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl...
, 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
Blue white screen
The blue-white screen is a screening technique that allows for the detection of successful ligations in vector-based gene cloning. DNA of interest is ligated into a vector. The vector is then transformed into competent cell . The competent cells are grown in the presence of X-gal...
. Its production may be induced by a non-hydrolyzable analog of allolactose
Allolactose
Allolactose is a disaccharide similar to lactose. It consists of the monosaccharides D-galactose and D-glucose linked through a β1-6 glycosidic linkage instead of the β1-4 linkage of lactose. It may arise from by the occasional transglycosylation of lactose by β-galactosidase.It is an inducer of...
, IPTG
IPTG
Isopropyl β-D-1-thiogalactopyranoside, abbreviated IPTG, is a molecular biology reagent.This compound is used as a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon...
, 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 accumulated in lysosomes in senescent cells, it is used as a senescence
Senescence
Senescence or biological aging is the change in the biology of an organism as it ages after its maturity. Such changes range from those affecting its cells and their function to those affecting the whole organism...
biomarker both in vivo and in vitro in qualitative and quantitative assays, despite its limitations.