Glucuronidase
Encyclopedia
Beta-glucuronidases are members of the glycosidase family of enzyme
s that catalyze breakdown of complex carbohydrates. Human β-glucuronidase is a type of glucuronidase (a member of glycosidase Family 2) that catalyzes hydrolysis
of β-D-glucuronic acid
residues from the non-reducing end of mucopolysaccharides
(also referred to as glycosaminoglycan
s) such as heparan sulfate
. Human β-glucuronidase is located in the lysosome
. In the gut, brush border β-glucuronidase converts conjugated bilirubin
to the unconjugated form for reabsorption. Beta-glucuronidase is also present in breast milk, and its deficiency contributes to neonatal jaundice
.
monomer
(653 amino acids) before proteolysis
removes 18 amino acids from the C-terminal end to form a 78 kDa monomer.
Beta-glucuronidase exists as a 332 kDa homotetramer. Beta-glucuronidase contains several notable structural formations, including a type of beta barrel
known as a jelly roll barrel and a TIM barrel
.
to the Escherichia coli
enzyme β-galactosidase. This homologous relationship, along with the knowledge that glycosidases often perform hydrolysis catalyzed by two acidic residues
, enabled the development of a mechanistic hypothesis. This hypothesis proposes that the two glutamic acid
residues Glu540 and Glu451 are the nucleophilic and acidic residues, respectively, and that the tyrosine
residue Tyr504 is also involved in catalysis.
In support of this hypothesis, experimental mutations in any of these three residues result in large decreases of enzymatic activity. Increased activity of an E451A mutant enzyme (where Glu451 is replaced with an alanine
residue) after addition of azide
is consistent with Glu451 as the acid/base residue. Using analysis of labeled β-glucuronidase peptides after hydrolysis of a substrate that enters a very stable intermediate stage, researchers have determined that Glu540 is the nucleophilic residue.
Though the particular type of nucleophilic substitution
employed by β-glucuronidase is unclear, evidence for the mechanisms of their homologues in the glycosidase family suggests that these reactions are qualitatively SN2 reactions. The reactions proceed through a transition state
with oxocarbenium ion characteristics. Initially, these mechanisms, because of this oxocarbenium characteristic of the transition state, were suggested to be SN1 reactions proceeding through a discrete oxocarbenium ion intermediate
. However, more recent evidence suggests that these oxocarbenium ion states have lifetimes of 10 femtoseconds - 0.1 nanoseconds (similar to that of a bond vibration period). These lifetimes are too short to assign to a reaction intermediate. From this evidence, it appears that these reactions, while having an SN1 appearance due to the oxocarbenium ion characteristics of their transition states, must be qualitatively SN2 reactions.
The specific activity of Tyr504 in the catalytic mechanism is unclear. Through comparison to the structural data of the homologous enzyme xylanase
, it has been suggested that Tyr504 of β-glucuronidase might stabilize the leaving nucleophile (Glu540) or modulate its activity.
In addition to these residues, a conserved asparagine
residue (Asn450) has been suggested to stabilize the substrate through the action of a hydrogen bond at the 2-hydroxyl group of the sugar substrate.
or Mucopolysaccharidosis
VII. A deficiency in this enzyme results in the build-up of non-hydrolyzed mucopolysaccharides in the patient. This disease can be extremely debilitating for the patient or can result in hydrops fetalis
prior to birth. In addition, mental retardation, short stature, coarse facial features, spinal abnormalities, and enlargement of liver and spleen are observed in surviving patients. This disease has been modeled in a strain of mice as well as a family of dogs. More recently researchers have discovered a feline family that exhibits deficiencies in β-glucuronidase activity. The source of this reduction of activity has been identified as an E351K mutation (Glu351 is mutated to a lysine residue). Glu351 is conserved in mammalian species, which suggests an important function for this residue. Examination of the human X-ray crystal
structure suggests that this residue (Glu352 in the human enzyme), which is buried deep within the TIM barrel
domain
, may be important for stabilization of the tertiary structure
of the enzyme. In the crystal structure, it appears that Arg216, a member of the jelly roll domain of the protein, forms a salt bridge
with Glu352; therefore, Glu352 is likely involved in stabilizing the interaction between two different three-dimensional domains of the enzyme.
, β-glucuronidase is used as a reporter gene
to monitor gene expression
in mammalian and plant cells. Monitoring β-glucuronidase activity through the use of a GUS assay allows determination of the spatial and temporal expression of the gene in question.
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...
s that catalyze breakdown of complex carbohydrates. Human β-glucuronidase is a type of glucuronidase (a member of glycosidase Family 2) that catalyzes 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-D-glucuronic acid
Glucuronic acid
Glucuronic acid is a carboxylic acid. Its structure is similar to that of glucose. However, glucuronic acid's sixth carbon is oxidized to a carboxylic acid...
residues from the non-reducing end of mucopolysaccharides
Glycosaminoglycan
Glycosaminoglycans or mucopolysaccharides are long unbranched polysaccharides consisting of a repeating disaccharide unit. The repeating unit consists of a hexose or a hexuronic acid, linked to a hexosamine .-Production:Protein cores made in the rough endoplasmic reticulum are posttranslationally...
(also referred to as glycosaminoglycan
Glycosaminoglycan
Glycosaminoglycans or mucopolysaccharides are long unbranched polysaccharides consisting of a repeating disaccharide unit. The repeating unit consists of a hexose or a hexuronic acid, linked to a hexosamine .-Production:Protein cores made in the rough endoplasmic reticulum are posttranslationally...
s) such as heparan sulfate
Heparan sulfate
Heparan sulfate is a linear polysaccharide found in all animal tissues. It occurs as a proteoglycan in which two or three HS chains are attached in close proximity to cell surface or extracellular matrix proteins...
. Human β-glucuronidase is located in the lysosome
Lysosome
thumb|350px|Schematic of typical animal cell, showing subcellular components. [[Organelle]]s: [[nucleoli]] [[cell nucleus|nucleus]] [[ribosomes]] [[vesicle |vesicle]] rough [[endoplasmic reticulum]]...
. In the gut, brush border β-glucuronidase converts conjugated bilirubin
Bilirubin
Bilirubin is the yellow breakdown product of normal heme catabolism. Heme is found in hemoglobin, a principal component of red blood cells. Bilirubin is excreted in bile and urine, and elevated levels may indicate certain diseases...
to the unconjugated form for reabsorption. Beta-glucuronidase is also present in breast milk, and its deficiency contributes to neonatal jaundice
Neonatal jaundice
Neonatal jaundice or Neonatal hyperbilirubinemia is a yellowing of the skin and other tissues of a newborn infant. A bilirubin level of more than 85 umol/l manifests clinical jaundice in neonates whereas in adults a level of 34 umol/l would look icteric...
.
Structure
Human β-glucuronidase is synthesized as an 80 kDaKDA
KDA may refer to:* Karachi Development Authority* Kongsberg Defence & Aerospace* Kotelawala Defence Academy* Kramer Design Associates* Lithium diisopropylamide, KDA is the potassium analogue of lithium diisopropylamideOr kDa may refer to:...
monomer
Monomer
A monomer is an atom or a small molecule that may bind chemically to other monomers to form a polymer; the term "monomeric protein" may also be used to describe one of the proteins making up a multiprotein complex...
(653 amino acids) before proteolysis
Proteolysis
Proteolysis is the directed degradation of proteins by cellular enzymes called proteases or by intramolecular digestion.-Purposes:Proteolysis is used by the cell for several purposes...
removes 18 amino acids from the C-terminal end to form a 78 kDa monomer.
Beta-glucuronidase exists as a 332 kDa homotetramer. Beta-glucuronidase contains several notable structural formations, including a type of beta 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...
known as a jelly roll barrel and a TIM 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...
.
Mechanism of catalysis
Human β-glucuronidase is homologousHomology (biology)
Homology forms the basis of organization for comparative biology. In 1843, Richard Owen defined homology as "the same organ in different animals under every variety of form and function". Organs as different as a bat's wing, a seal's flipper, a cat's paw and a human hand have a common underlying...
to the Escherichia 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...
enzyme β-galactosidase. This homologous relationship, along with the knowledge that glycosidases often perform hydrolysis catalyzed by two acidic residues
Residue (chemistry)
In chemistry, residue is the material remaining after a distillation or an evaporation, or to a portion of a larger molecule, such as a methyl group. It may also refer to the undesired byproducts of a reaction....
, enabled the development of a mechanistic hypothesis. This hypothesis proposes that the two 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...
residues Glu540 and Glu451 are the nucleophilic and acidic residues, respectively, and that the tyrosine
Tyrosine
Tyrosine or 4-hydroxyphenylalanine, is one of the 22 amino acids that are used by cells to synthesize proteins. Its codons are UAC and UAU. It is a non-essential amino acid with a polar side group...
residue Tyr504 is also involved in catalysis.
In support of this hypothesis, experimental mutations in any of these three residues result in large decreases of enzymatic activity. Increased activity of an E451A mutant enzyme (where Glu451 is replaced with an alanine
Alanine
Alanine is an α-amino acid with the chemical formula CH3CHCOOH. The L-isomer is one of the 20 amino acids encoded by the genetic code. Its codons are GCU, GCC, GCA, and GCG. It is classified as a nonpolar amino acid...
residue) after addition of azide
Azide
Azide is the anion with the formula N3−. It is the conjugate base of hydrazoic acid. N3− is a linear anion that is isoelectronic with CO2 and N2O. Per valence bond theory, azide can be described by several resonance structures, an important one being N−=N+=N−...
is consistent with Glu451 as the acid/base residue. Using analysis of labeled β-glucuronidase peptides after hydrolysis of a substrate that enters a very stable intermediate stage, researchers have determined that Glu540 is the nucleophilic residue.
Though the particular type of nucleophilic substitution
Nucleophilic substitution
In organic and inorganic chemistry, nucleophilic substitution is a fundamental class of reactions in which an electron nucleophile selectively bonds with or attacks the positive or partially positive charge of an atom or a group of atoms called the leaving group; the positive or partially positive...
employed by β-glucuronidase is unclear, evidence for the mechanisms of their homologues in the glycosidase family suggests that these reactions are qualitatively SN2 reactions. The reactions proceed through a transition state
Transition state
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 energy along this reaction coordinate. At this point, assuming a perfectly irreversible reaction, colliding reactant molecules will always...
with oxocarbenium ion characteristics. Initially, these mechanisms, because of this oxocarbenium characteristic of the transition state, were suggested to be SN1 reactions proceeding through a discrete oxocarbenium ion intermediate
Reaction intermediate
A reaction intermediate or an intermediate is a molecular entity that is formed from the reactants and reacts further to give the directly observed products of a chemical reaction. Most chemical reactions are stepwise, that is they take more than one elementary step to complete...
. However, more recent evidence suggests that these oxocarbenium ion states have lifetimes of 10 femtoseconds - 0.1 nanoseconds (similar to that of a bond vibration period). These lifetimes are too short to assign to a reaction intermediate. From this evidence, it appears that these reactions, while having an SN1 appearance due to the oxocarbenium ion characteristics of their transition states, must be qualitatively SN2 reactions.
The specific activity of Tyr504 in the catalytic mechanism is unclear. Through comparison to the structural data of the homologous enzyme xylanase
Xylanase
Xylanase is the name given to a class of enzymes which degrade the linear polysaccharide beta-1,4-xylan into xylose, thus breaking down hemicellulose, one of the major components of plant cell walls....
, it has been suggested that Tyr504 of β-glucuronidase might stabilize the leaving nucleophile (Glu540) or modulate its activity.
In addition to these residues, a conserved asparagine
Asparagine
Asparagine is one of the 20 most common natural amino acids on Earth. It has carboxamide as the side-chain's functional group. It is not an essential amino acid...
residue (Asn450) has been suggested to stabilize the substrate through the action of a hydrogen bond at the 2-hydroxyl group of the sugar substrate.
Sly Syndrome
Deficiencies in β-glucuronidase result in the non recessive inherited metabolic disease known as Sly syndromeSly syndrome
Sly syndrome, also called Mucopolysaccharidosis Type VII or MPS, is an autosomal recessive lysosomal storage disease characterized by a deficiency of the enzyme β-glucuronidase, a lysosomal enzyme. Sly syndrome belongs to a group of disorders known as mucopolysaccharidoses, which are lysosomal...
or Mucopolysaccharidosis
Mucopolysaccharidosis
Mucopolysaccharidoses are a group of metabolic disorders caused by the absence or malfunctioning of lysosomal enzymes needed to break down molecules called glycosaminoglycans - long chains of sugar carbohydrates in each of our cells that help build bone, cartilage, tendons, corneas, skin and...
VII. A deficiency in this enzyme results in the build-up of non-hydrolyzed mucopolysaccharides in the patient. This disease can be extremely debilitating for the patient or can result in hydrops fetalis
Hydrops fetalis
Hydrops fetalis is a condition in the fetus characterized by an accumulation of fluid, or edema, in at least two fetal compartments. By comparison, hydrops allantois or hydrops amnion are an accumulation of excessive fluid in the allantoic or amniotic space respectively.-Presentation:Locations can...
prior to birth. In addition, mental retardation, short stature, coarse facial features, spinal abnormalities, and enlargement of liver and spleen are observed in surviving patients. This disease has been modeled in a strain of mice as well as a family of dogs. More recently researchers have discovered a feline family that exhibits deficiencies in β-glucuronidase activity. The source of this reduction of activity has been identified as an E351K mutation (Glu351 is mutated to a lysine residue). Glu351 is conserved in mammalian species, which suggests an important function for this residue. Examination of the human X-ray crystal
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
structure suggests that this residue (Glu352 in the human enzyme), which is buried deep within the TIM 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...
domain
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...
, may be important for stabilization of the tertiary structure
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...
of the enzyme. In the crystal structure, it appears that Arg216, a member of the jelly roll domain of the protein, forms a salt bridge
Salt bridge (protein)
Salt bridges fall into the broader category of noncovalent interactions. A salt bridge is actually a combination of two noncovalent interactions: hydrogen bonding and electrostatic interactions . This is most commonly observed to contribute stability to the entropically unfavorable folded...
with Glu352; therefore, Glu352 is likely involved in stabilizing the interaction between two different three-dimensional domains of the enzyme.
Molecular Applications: Use as a Reporter Gene
In molecular biologyMolecular 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...
, β-glucuronidase is used as a reporter gene
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...
to monitor gene expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
in mammalian and plant cells. Monitoring β-glucuronidase activity through the use of a GUS assay allows determination of the spatial and temporal expression of the gene in question.
- Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081).
See also
- Alpha-glucuronidaseAlpha-glucuronidaseFor beta-glucuronidase, see Beta-glucuronidaseIn enzymology, an alpha-glucuronidase is an enzyme that catalyzes the chemical reactionThus, the two substrates of this enzyme are alpha-D-glucuronoside and H2O, whereas its two products are alcohol and D-glucuronate.This enzyme belongs to the family...
- Glucuronosyl-disulfoglucosamine glucuronidaseGlucuronosyl-disulfoglucosamine glucuronidaseIn enzymology, a glucuronosyl-disulfoglucosamine glucuronidase is an enzyme that catalyzes the chemical reactionThus, the two substrates of this enzyme are 3-D-glucuronosyl-N2,6-disulfo-beta-D-glucosamine and H2O, whereas its two products are D-glucuronate and N2,6-disulfo-D-glucosamine.This...
- Glycyrrhizinate beta-glucuronidaseGlycyrrhizinate beta-glucuronidaseIn enzymology, a glycyrrhizinate beta-glucuronidase is an enzyme that catalyzes the chemical reactionThus, the two substrates of this enzyme are glycyrrhizinate and H2O, whereas its two products are 1,2-beta-D-glucuronosyl-D-glucuronate and glycyrrhetinate....
External links
- Updated research on reporter glucuronidase and other reporters from Reportergene
- Database of Catalytic Mechanism Research and other information on beta-glucuronidase