CAZypedia needs your help! We have many unassigned GH, PL, CE, AA, GT, and CBM pages in need of Authors and Responsible Curators.
Scientists at all career stages, including students, are welcome to contribute to CAZypedia. Read more here, and in the 10th anniversary article in Glycobiology.
New to the CAZy classification? Read this first.
Consider attending the 15th Carbohydrate Bioengineering Meeting in Ghent, 5-8 May 2023.
Glycoside Hydrolase Family 2
This page has been approved by the Responsible Curator as essentially complete. CAZypedia is a living document, so further improvement of this page is still possible. If you would like to suggest an addition or correction, please contact the page's Responsible Curator directly by e-mail.
|Glycoside Hydrolase Family GH2|
|Active site residues||known|
|CAZy DB link|
The most common activities for glycoside hydrolases of this family include β-galactosidases, β-glucuronidases, β-mannosidases, exo-β-glucosaminidases and, in plants, a mannosylglycoprotein endo-β-mannosidase. The enzymes are found across a broad spectrum of life forms, but are concentrated in bacteria. The most famous enzyme in this family is the E. coli (lacZ) β-galactosidase, a component of the lac operon. Not only did this enzyme play a key role in developing the understanding of operon structure and control of gene expression, but also it continues to play a key role as a cell biological probe. Another matter of note is that this remains the largest protein monomer to be sequenced entirely at the peptide level . E. coli also contains a second, vestigial β-galactosidase (ebg) whose activity has been shown to evolve in lacZ- strains of E. coli grown under selective pressure with lactose as sole carbon source [2, 3]. Another reasonably well-studied GH2 enzyme is the E. coli β-glucuronidase, whose activity is used to detect the presence of E. coli (http://www.cfsan.fda.gov/~ebam/bam-4.html), though interestingly not the nasty O157 strain. The principal enzyme of medical interest in GH2 is the lysosomal β-glucuronidase whose deficiency leads to Sly syndrome . The only other human GH2 enzyme is the lysosomal β-mannosidase.
Kinetics and Mechanism
Family 2 β-glycosidases are retaining enzymes and follow a classical Koshland double-displacement mechanism. This was first evidenced in 1951 by Wallenfels, who demonstrated the transglycosylation of lactose via an implicated glycosyl-enzyme intermediate  The best studied enzyme kinetically must be the E. coli (lacZ) β-galactosidase, for which a key set of studies defining the two-step mechanism and elucidating rate-limiting steps was published by the groups of Yon and Sinnott in the early 1970’s [6, 7, 8]. Indeed the approaches developed on that system laid the foundations for many subsequent studies on other glycosidases. An analysis of the roles of each substrate hydroxyl in catalysis, based upon kinetic studies with modified sugars has also been published . Some GH2 glycosidases require Mg2+ for activity and in E. coli β-galactosidase this Mg2+ requirement is associated with the binding of the cation in the active site such that it places the acid/base residue appropriately. Others, such as the human β-glucuronidase, have no such metal ion requirement.
The catalytic nucleophile in GH2 was first correctly identified in the E. coli (lacZ) β-galactosidase as Glu537 in the sequence ILCEYAH through trapping of the 2-deoxy-2-fluorogalactosyl-enzyme intermediate and subsequent peptide mapping via HPLC techniques using radiolabeled tracers . Earlier studies, carefully done using conduritol C cis-epoxide as affinity label, had identified Glu461 as the labeled residue,  on which basis a series of beautifully executed kinetic studies were performed on mutants modified at this position that appeared initially to support this conclusion . However, doubts were raised when similar kinetic analysis of nucleophile mutants of the GH1 Agrobacterium sp. β-glucosidase yielded quite different results, leading to the above labeling study . The general acid/base catalyst was then identified as Glu461 by re-interpretation  of the published kinetic results on mutants at that position , which had included azide rescue experiments. These conclusions were fully supported by subsequent 3-dimensional structural analyses (below).
Three-dimensional structures are available for five Family GH2 enzymes currently, the first solved being that of the E. coli (lacZ) β-galactosidase in a tour de force of X-ray crystallography at that time, given its huge size (4 x 125,000 Da) . The enzyme is multidomain, but as members of Clan GHA the catalytic domain is a classical (α/β)8 TIM barrel fold with the two key active site glutamic acids being approximately 200 residues apart in sequence and located at the C-terminal ends of β-strands 4 (acid/base) and 7 (nucleophile).
- First stereochemistry determination
- Via transglycosylation of lactose 
- First catalytic nucleophile identification
- E. coli (lacZ) β-galactosidase by 2-fluorogalactose labeling 
- First general acid/base residue identification
- E. coli (lacZ) β-galactosidase by re-interpretation of kinetic studies with mutants [10, 12]
- First 3-D structure
- E. coli (lacZ) β-galactosidase 
- Fowler AV and Zabin I. (1978). Amino acid sequence of beta-galactosidase. XI. Peptide ordering procedures and the complete sequence. J Biol Chem. 1978;253(15):5521-5. | Google Books | Open Library
- Hall BG (1999). Experimental evolution of Ebg enzyme provides clues about the evolution of catalysis and to evolutionary potential. FEMS Microbiol Lett. 1999;174(1):1-8. DOI:10.1111/j.1574-6968.1999.tb13542.x |
- Krishnan S, Hall BG, and Sinnott ML. (1995). Catalytic consequences of experimental evolution: catalysis by a 'third-generation' evolvant of the second beta-galactosidase of Escherichia coli, ebgabcde, and by ebgabcd, a 'second-generation' evolvant containing two supposedly 'kinetically silent' mutations. Biochem J. 1995;312 ( Pt 3)(Pt 3):971-7. DOI:10.1042/bj3120971 |
- Sly WS, Quinton BA, McAlister WH, and Rimoin DL. (1973). Beta glucuronidase deficiency: report of clinical, radiologic, and biochemical features of a new mucopolysaccharidosis. J Pediatr. 1973;82(2):249-57. DOI:10.1016/s0022-3476(73)80162-3 |
Wallenfels K. Enzymatische synthese von oligosacchariden aus disacchariden. Naturwissenschaften, 1951; 38: 306-307. DOI:10.1007/BF00636782
- Sinnott ML and Souchard IJ. (1973). The mechanism of action of beta-galactosidase. Effect of aglycone nature and -deuterium substitution on the hydrolysis of aryl galactosides. Biochem J. 1973;133(1):89-98. DOI:10.1042/bj1330089 |
- Sinnott ML and Viratelle OM. (1973). The effect of methanol and dioxan on the rates of the beta-galactosidase-catalysed hydrolyses of some beta-D-galactrophyranosides: rate-limiting degalactosylation. The ph-dependence of galactosylation and degalactosylation. Biochem J. 1973;133(1):81-7. DOI:10.1042/bj1330081 |
- Viratelle OM and Yon JM. (1973). Nucleophilic competition in some -galactosidase-catalyzed reactions. Eur J Biochem. 1973;33(1):110-6. DOI:10.1111/j.1432-1033.1973.tb02661.x |
- McCarter JD, Adam MJ, and Withers SG. (1992). Binding energy and catalysis. Fluorinated and deoxygenated glycosides as mechanistic probes of Escherichia coli (lacZ) beta-galactosidase. Biochem J. 1992;286 ( Pt 3)(Pt 3):721-7. DOI:10.1042/bj2860721 |
- Gebler JC, Aebersold R, and Withers SG. (1992). Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) beta-galactosidase from Escherichia coli. J Biol Chem. 1992;267(16):11126-30. | Google Books | Open Library
- Herrchen M and Legler G. (1984). Identification of an essential carboxylate group at the active site of lacZ beta-galactosidase from Escherichia coli. Eur J Biochem. 1984;138(3):527-31. DOI:10.1111/j.1432-1033.1984.tb07947.x |
- Cupples CG, Miller JH, and Huber RE. (1990). Determination of the roles of Glu-461 in beta-galactosidase (Escherichia coli) using site-specific mutagenesis. J Biol Chem. 1990;265(10):5512-8. | Google Books | Open Library
- Jacobson RH, Zhang XJ, DuBose RF, and Matthews BW. (1994). Three-dimensional structure of beta-galactosidase from E. coli. Nature. 1994;369(6483):761-6. DOI:10.1038/369761a0 |