Î’-Galactosidase
   HOME

TheInfoList



OR:

β-Galactosidase (EC 3.2.1.23, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase) is a
glycoside hydrolase In biochemistry, glycoside hydrolases (also called glycosidases or glycosyl hydrolases) are a class of enzymes which catalysis, catalyze the hydrolysis of glycosidic bonds in polysaccharide, complex sugars. They are extremely common enzymes, wi ...
enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...
that
catalyzes Catalysis () is the increase in rate of a chemical reaction due to an added substance known as a catalyst (). Catalysts are not consumed by the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recycles quick ...
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 Lactase () is an enzyme produced by many organisms and is essential to the complete digestion of whole milk. It breaks down the sugar lactose into its component parts, galactose and glucose. Lactase is found in the brush border of the small ...
but that name is generally reserved for mammalian digestive enzymes that breaks down lactose specifically.) β-Galactosides include carbohydrates containing
galactose Galactose (, ''wikt:galacto-, galacto-'' + ''wikt:-ose#Suffix 2, -ose'', ), sometimes abbreviated Gal, is a monosaccharide sugar that is about as sweetness, sweet as glucose, and about 65% as sweet as sucrose. It is an aldohexose and a C-4 epime ...
where the glycosidic bond lies above the galactose molecule.
Substrate Substrate may refer to: Physical layers *Substrate (biology), the natural environment in which an organism lives, or the surface or medium on which an organism grows or is attached ** Substrate (aquatic environment), the earthy material that exi ...
s of different β-galactosidases include
ganglioside A ganglioside is a molecule composed of a glycosphingolipid (ceramide and oligosaccharide) with one or more sialic acids (e.g. N-acetylneuraminic acid, ''N''-acetylneuraminic acid, NANA) linked on the sugar chain. NeuNAc, an acetylated derivative ...
GM1, lactosylceramides,
lactose Lactose is a disaccharide composed of galactose and glucose and has the molecular formula C12H22O11. Lactose makes up around 2–8% of milk (by mass). The name comes from (Genitive case, gen. ), the Latin word for milk, plus the suffix ''-o ...
, and various
glycoprotein Glycoproteins are proteins which contain oligosaccharide (sugar) chains covalently attached to amino acid side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known a ...
s.


Function

β-Galactosidase is an exoglycosidase which hydrolyzes the β-
glycosidic bond A glycosidic bond or glycosidic linkage is a type of ether bond that joins a carbohydrate (sugar) molecule to another group, which may or may not be another carbohydrate. A glycosidic bond is formed between the hemiacetal or hemiketal group o ...
formed between a
galactose Galactose (, ''wikt:galacto-, galacto-'' + ''wikt:-ose#Suffix 2, -ose'', ), sometimes abbreviated Gal, is a monosaccharide sugar that is about as sweetness, sweet as glucose, and about 65% as sweet as sucrose. It is an aldohexose and a C-4 epime ...
and its organic moiety. It may also cleave fucosides and
arabinosides Arabinose is an aldopentose – a monosaccharide containing five carbon atoms, and including an aldehyde (CHO) functional group. Properties For biosynthetic reasons, most saccharides are almost always more abundant in nature as the "D"-form, or ...
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 ''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escherichia'' that is commonly foun ...
'', 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 Lactose is a disaccharide composed of galactose and glucose and has the molecular formula C12H22O11. Lactose makes up around 2–8% of milk (by mass). The name comes from (Genitive case, gen. ), the Latin word for milk, plus the suffix ''-o ...
when
glucose Glucose is a sugar with the Chemical formula#Molecular formula, molecular formula , which is often abbreviated as Glc. It is overall the most abundant monosaccharide, a subcategory of carbohydrates. It is mainly made by plants and most algae d ...
level is low. β-Galactosidase synthesis stops when glucose levels are sufficient. 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. 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. 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 Lactase () is an enzyme produced by many organisms and is essential to the complete digestion of whole milk. It breaks down the sugar lactose into its component parts, galactose and glucose. Lactase is found in the brush border of the small ...
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.


Structure

The 1,023
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...
s of ''
E. coli ''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escherichia'' that is commonly foun ...
'' β-galactosidase were sequenced in 1983, and its structure determined eleven years later in 1994. The
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...
is a 464-
kDa The dalton or unified atomic mass unit (symbols: Da or u, respectively) is a unit of mass defined as of the mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state and at rest. It is a non-SI unit accepted f ...
homotetramer A tetrameric protein is a protein with a quaternary structure of four subunits (tetrameric). Homotetramers have four identical subunits (such as glutathione S-transferase), and heterotetramers are complexes of different subunits. A tetramer ...
with 2,2,2-point
symmetry Symmetry () in everyday life refers to a sense of harmonious and beautiful proportion and balance. In mathematics, the term has a more precise definition and is usually used to refer to an object that is Invariant (mathematics), invariant und ...
. Each unit of β-galactosidase consists of five domains; domain 1 is a jelly-roll type
β-barrel In protein structures, a beta barrel (β barrel) is a beta sheet (β sheet) composed of Protein tandem repeats, tandem repeats that twists and coils to form a closed toroidal structure in which the first strand is bonded to the last strand (hydrog ...
, 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. 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

β-Galactosidase can catalyze three different reactions in organisms. In one, it can go through a process called transgalactosylation to make
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 the occasional transglycosylation ...
, creating a
positive feedback loop Positive feedback (exacerbating feedback, self-reinforcing feedback) is a process that occurs in a feedback loop where the outcome of a process reinforces the inciting process to build momentum. As such, these forces can exacerbate the effects ...
for the production of β-galactose. Allolactose can also be cleaved to form monosaccharides. It can also hydrolyze lactose into
galactose Galactose (, ''wikt:galacto-, galacto-'' + ''wikt:-ose#Suffix 2, -ose'', ), sometimes abbreviated Gal, is a monosaccharide sugar that is about as sweetness, sweet as glucose, and about 65% as sweet as sucrose. It is an aldohexose and a C-4 epime ...
and
glucose Glucose is a sugar with the Chemical formula#Molecular formula, molecular formula , which is often abbreviated as Glc. It is overall the most abundant monosaccharide, a subcategory of carbohydrates. It is mainly made by plants and most algae d ...
which will proceed into
glycolysis Glycolysis is the metabolic pathway that converts glucose () into pyruvic acid, pyruvate and, in most organisms, occurs in the liquid part of cells (the cytosol). The Thermodynamic free energy, free energy released in this process is used to form ...
. The active site of β-galactosidase catalyzes the hydrolysis of its
disaccharide A disaccharide (also called a double sugar or ''biose'') is the sugar formed when two monosaccharides are joined by glycosidic linkage. Like monosaccharides, disaccharides are simple sugars soluble in water. Three common examples are sucrose, ...
substrate via "shallow" (nonproductive site) and "deep" (productive site) binding.
Galactoside A galactoside is a glycoside containing galactose. The H of the OH group on carbon-1 of galactose is replaced by an organic moiety. Depending on whether the glycosidic bond lies "above" or "below" the plane of the galactose molecule, galactosides ...
s such as PETG and IPTG will bind in the shallow site when the enzyme is in "open" conformation while
transition state analog Transition state analogs (transition state analogues), are chemical compounds with a chemical structure that resembles the transition state of a substrate molecule in an enzyme-catalyzed chemical reaction. Enzymes interact with a substrate by mea ...
s such as L-ribose and D-galactonolactone will bind in the deep site when the conformation is "closed". 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 state In chemistry, the transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest potential energy along this reaction coordinate. It is often marked w ...
s 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 . It has been kinetically measured that single tetramers of the protein catalyze reactions at a rate of 38,500 ± 900 reactions per minute. Monovalent
potassium Potassium is a chemical element; it has Symbol (chemistry), symbol K (from Neo-Latin ) and atomic number19. It is a silvery white metal that is soft enough to easily cut with a knife. Potassium metal reacts rapidly with atmospheric oxygen to ...
ion An ion () is an atom or molecule with a net electrical charge. The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by convent ...
s (K+) as well as divalent
magnesium Magnesium is a chemical element; it has Symbol (chemistry), symbol Mg and atomic number 12. It is a shiny gray metal having a low density, low melting point and high chemical reactivity. Like the other alkaline earth metals (group 2 ...
ions (Mg2+) are required for the enzyme's optimal activity. The β-linkage of the substrate is cleaved by a terminal
carboxyl In organic chemistry, a carboxylic acid is an organic acid that contains a carboxyl group () attached to an R-group. The general formula of a carboxylic acid is often written as or , sometimes as with R referring to an organyl group (e.g. ...
group on the
side chain In organic chemistry and biochemistry, a side chain is a substituent, chemical group that is attached to a core part of the molecule called the "main chain" or backbone chain, backbone. The side chain is a hydrocarbon branching element of a mo ...
of a
glutamic acid Glutamic acid (symbol Glu or E; known as glutamate in its anionic form) is an α- amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a non-essential nutrient for humans, meaning that the human body can ...
. In ''
E. coli ''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escherichia'' that is commonly foun ...
'', Glu-461 was thought to be the
nucleophile In chemistry, a nucleophile is a chemical species that forms bonds by donating an electron pair. All molecules and ions with a free pair of electrons or at least one pi bond can act as nucleophiles. Because nucleophiles donate electrons, they are ...
in the
substitution Substitution may refer to: Arts and media *Substitution (poetry), a variation in poetic scansion * Substitution (theatre), an acting methodology Music *Chord substitution, swapping one chord for a related one within a chord progression *Tritone ...
reaction. However, it is now known that Glu-461 is an
acid An acid is a molecule or ion capable of either donating a proton (i.e. Hydron, hydrogen cation, H+), known as a Brønsted–Lowry acid–base theory, Brønsted–Lowry acid, or forming a covalent bond with an electron pair, known as a Lewis ...
catalyst. Instead, Glu-537 is the actual nucleophile, binding to a galactosyl intermediate. In
human Humans (''Homo sapiens'') or modern humans are the most common and widespread species of primate, and the last surviving species of the genus ''Homo''. They are Hominidae, great apes characterized by their Prehistory of nakedness and clothing ...
s, the
nucleophile In chemistry, a nucleophile is a chemical species that forms bonds by donating an electron pair. All molecules and ions with a free pair of electrons or at least one pi bond can act as nucleophiles. Because nucleophiles donate electrons, they are ...
of the
hydrolysis Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...
reaction is Glu-268. 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.


Applications

The β-galactosidase assay is used frequently in
genetics Genetics is the study of genes, genetic variation, and heredity in organisms.Hartl D, Jones E (2005) It is an important branch in biology because heredity is vital to organisms' evolution. Gregor Mendel, a Moravian Augustinians, Augustinian ...
,
molecular biology Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactio ...
, and other
life science Life, also known as biota, refers to matter that has biological processes, such as signaling and self-sustaining processes. It is defined descriptively by the capacity for homeostasis, organisation, metabolism, growth, adaptation, respon ...
s. An active enzyme may be detected using artificial chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside,
X-gal X-gal (also abbreviated BCIG for 5-bromo-4-chloro-3-indolyl-β--galactopyranoside) is an organic compound consisting of galactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and collaborators in 1964. The formal ...
. β-galactosidase will cleave the glycosidic bond in
X-gal X-gal (also abbreviated BCIG for 5-bromo-4-chloro-3-indolyl-β--galactopyranoside) is an organic compound consisting of galactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and collaborators in 1964. The formal ...
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. It is used for example in
blue white screen Blue is one of the three primary colours in the RYB colour model (traditional colour theory), as well as in the RGB (additive) colour model. It lies between violet and cyan on the spectrum of visible light. The term ''blue'' generally des ...
. Its production may be induced by a non-hydrolyzable
analog Analog or analogue may refer to: Computing and electronics * Analog signal, in which information is encoded in a continuous variable ** Analog device, an apparatus that operates on analog signals *** Analog electronics, circuits which use analog ...
of
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 the occasional transglycosylation ...
, 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, LacZ α and LacZ Ω, 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 A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, o ...
s where the presence of the ''lacZα'' gene in a plasmid can complement in ''
trans Trans- is a Latin prefix meaning "across", "beyond", or "on the other side of". Used alone, trans may refer to: Sociology * Trans, a sociological term which may refer to: ** Transgender, people who identify themselves with a gender that di ...
'' 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 (also abbreviated BCIG for 5-bromo-4-chloro-3-indolyl-β--galactopyranoside) is an organic compound consisting of galactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and collaborators in 1964. The formal ...
, 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, exploiting β-galactosidase's tendency to oligomerise to suggest a potential role for oligomericity in MLL fusion protein function. A recent study conducted in 2020–2021 determined that β-Galactosidase activity correlates with
cellular senescence Cellular senescence is a phenomenon characterized by the cessation of cell division. In their experiments during the early 1960s, Leonard Hayflick and Paul Moorhead found that normal human fetal fibroblasts in culture reach a maximum of appro ...
. Cellular senescence is the process where cells do not divide, but do not die either. In senescent cells β-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 senescent cells can be achieved by measuring the lysosomal β-Galactosidase activity. A particular isoform of β-Galactosidase exclusively expressed by senescent cells was hypothesized to exist because only these types of cells showed β-Galactosidase activity at pH 6.0 (Senescence Associated beta-gal or SA-beta-gal) . Specific quantitative assays were even developed for its detection. However, it is now known that this phenomenon occurs due to an overexpression and accumulation of the lysosomal endogenous β-Galactosidase, and thus it doesn't represent a distinct isoform of β-Galactosidase, nor is it a required phenotype for the progression or maintenance of the senescent state. 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. and, as a result, the mutant enzyme is able to replace the lacZ β-galactosidase. EbgA and LacZ are 50% identical on the DNA level and 33% identical on the amino acid level. 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 ''α''4 β4 hetero-octamer.


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. β-galactosidase genes can differ in the length of their coding sequence and the length of proteins formed by amino acids. This separates the β-galactosidases into four families: GHF-1, GHF-2, GHF-35, and GHF- 42. ''E. Coli'' belongs to GHF-2, all plants belong to GHF-35, and ''
Thermus thermophilus ''Thermus thermophilus'' is a gram stain, Gram-negative bacterium used in a range of biotechnological applications, including as a model organism for genetic manipulation, structural genomics, and systems biology. The bacterium is extremely therm ...
'' belongs to GHF-42. 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%. A study targeted at identifying fruit softening of peaches found 17 different gene expressions of β-galactosidases. The only other known crystal structure of β-galactosidase is from ''
Thermus thermophilus ''Thermus thermophilus'' is a gram stain, Gram-negative bacterium used in a range of biotechnological applications, including as a model organism for genetic manipulation, structural genomics, and systems biology. The bacterium is extremely therm ...
.''


References


External links

* {{DEFAULTSORT:Beta-Galactosidase Carbohydrate metabolism EC 3.2.1 Hydrolases Enzymes Aging-related enzymes Enzymes of known structure Lactose