Glycoside Hydrolase Family 2

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• Author: Stephen Withers
• Responsible Curator: Stephen Withers

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Glycoside Hydrolase Family GH2
Clan	GH-A
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/GH2.html

Substrate specificities

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 [1]. 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 [4]. 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 [5] 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 [9]. 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.

Catalytic Residues

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 [10]. Earlier studies, carefully done using conduritol C cis-epoxide as affinity label, had identified Glu461 as the labeled residue, [11] 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 [12]. 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 [10]. The general acid/base catalyst was then identified as Glu461 by re-interpretation [10] of the published kinetic results on mutants at that position [12], which had included azide rescue experiments. These conclusions were fully supported by subsequent 3-dimensional structural analyses (below).

Three-dimensional structures

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) [13]. The enzyme is multidomain, but as members of Clan GHA the catalytic domains 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).

Family Firsts

First stereochemistry determination

Via transglycosylation of lactose [5]

First catalytic nucleophile identification

E. coli (lacZ) β-galactosidase by 2-fluorogalactose labeling [10]

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 [13]

References

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5. Wallenfels K. Enzymatische Synthese von Oligosacchariden aus Disacchariden, Naturwissenschaften, 1951, 38, 306-307. doi:10.1007/BF00636782