Β-Glucuronidase

Structure

Human β-glucuronidase is synthesized as an 80 kDa monomer (653 amino acids) before proteolysis removes 18 amino acids from the C-terminal end to form a 78 kDa monomer. β-Glucuronidase exists as a 332 kDa homotetramer. β-Glucuronidase contains several notable structural formations, including a type of β-barrel known as a jelly roll barrel and a TIM barrel.

Mechanism of catalysis

Human β-glucuronidase is homologous 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. | Image:Heparan sulfate.JPG|Repeating unit of the heparan sulfate substrate of β-glucuronidase | Image:Beta Glucuronidase Active Site Pocket.jpg|Surface depiction of active site pocket of β-glucuronidase with catalytic residues shown | Image:Mechanism Of Beta Glucuronidase.JPG|Mechanism of β-glucuronidase hydrolysis of a sugar substrate with high energy transition states showing oxocarbenium ion character depicted | Image:File-Predicted Tyr 504 activity in Beta Glucuronidase.JPG|Potential stabilization of the nucleophilic residue Glu540 by Tyr504 in β-glucuronidase | Image:Predicted Asn 450 activity in beta glucuronidase.JPG|Predicted activity of the conserved Asn450 residue in stabilization of the β-glucuronidase sugar substrate | Image:Possible Salt Bridge Human Beta Glucuronidase.jpg|Potential salt bridge between Glu352 and Arg216 in human beta-glucuronidase

Sly syndrome

Main article: Sly syndrome

Deficiencies in β-glucuronidase result in the autosomal recessive inherited metabolic disease known as Sly syndrome 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. 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.

Use as a reporter gene

In molecular biology, 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.

• Molecular graphics images were produced using the Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco.

References

References

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