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.
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.
Beta-glucuronidase