Participation of asparagine 370 and glutamine 235 in the catalysis by acid beta-glucosidase: the enzyme deficient in Gaucher disease.

Abstract

The hydrolysis of glucosylceramide by acid beta-glucosidase proceeds via a two-step, double displacement mechanism that includes cleavage of the O-beta-glucosidic bond, enzyme-glucosylation and, then, enzyme-deglucosylation. Two residues that may impact this cycle are N370 and E235. The N370S mutant enzyme is very common in Gaucher disease type 1 patients. Homology and crystal data predictions suggested that E235 is the acid/base catalyst in the hydrolytic reaction. Here, the roles of N370 and E235 in hydrolysis were explored using mutant proteins with selected amino acid substitutions. Heterologously expressed enzymes were characterized using inhibitors, activators, and alternative substrates to gain insight into the effects on the glucosylation (single turnover) and deglucosylation (transglucosylation) steps in catalysis. Specific substitutions at N370 selectively altered only the glucosylation step whereas N370S altered this and the deglucosylation steps. To provide functional data to support E235 as the acid/base catalyst, progress curves with poor substrates with more acidic leaving groups were used in the presence and absence of azide as an exogenous nucleophile. The restoration of E235G activity to nearly wild-type levels was achieved using azide with 2,4-dinitrophenyl-beta-glucoside as substrate. The loss of the acidic arm of the pH optimum activity curve of E235G provided additional functional support for E235 as the acid/base in catalysis. This study provides insight into the function of these residues in acid beta-glucosidase active site function.

• Authors: Benjamin Liou, Gregory A Grabowski
• Journal: Molecular genetics and metabolism (Mol Genet Metab) Vol. 97 Issue 1 Pg. 65-74 (May 2009) ISSN: 1096-7206 [Electronic] United States
• PMID: 19217815 (Publication Type: Journal Article)

Chemical References

• Azides
• Mutant Proteins
• Phosphatidylserines
• Glutamine
• Taurocholic Acid
• Asparagine
• Glucosylceramidase
• 2,4-Dinitrophenol

Topics

• 2,4-Dinitrophenol
• Amino Acid Substitution (drug effects)
• Asparagine (metabolism)
• Azides (pharmacology)
• Biocatalysis (drug effects)
• Catalytic Domain
• Enzyme Activation (drug effects)
• Gaucher Disease (enzymology)
• Glucosylceramidase (antagonists & inhibitors, deficiency, metabolism)
• Glutamine (metabolism)
• Glycosylation (drug effects)
• Hydrogen-Ion Concentration (drug effects)
• Hydrolysis (drug effects)
• Mutant Proteins (metabolism)
• Phosphatidylserines (pharmacology)
• Structure-Activity Relationship
• Substrate Specificity (drug effects)
• Taurocholic Acid (pharmacology)