Glycoside Hydrolase Family 2

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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

Contents

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, as first shown by XXXXX [5] and follow a classical Koshland double-displacement mechanism. 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
(pmid=TBA)
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

  1. Fowler AV and Zabin I. Amino acid sequence of beta-galactosidase. XI. Peptide ordering procedures and the complete sequence. J Biol Chem. 1978 Aug 10;253(15):5521-5. PubMed ID:97298 | HubMed [FowlerZabin1978]
  2. Hall BG. Experimental evolution of Ebg enzyme provides clues about the evolution of catalysis and to evolutionary potential. FEMS Microbiol Lett. 1999 May 1;174(1):1-8. PubMed ID:10234816 | HubMed [Hall1999]
  3. Krishnan S, Hall BG, and Sinnott ML. 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 Dec 15;312 ( Pt 3):971-7. PubMed ID:8554546 | HubMed [Krishnan1995]
  4. Sly WS, Quinton BA, McAlister WH, and Rimoin DL. Beta glucuronidase deficiency: report of clinical, radiologic, and biochemical features of a new mucopolysaccharidosis. J Pediatr. 1973 Feb;82(2):249-57. PubMed ID:4265197 | HubMed [Sly1973]
  5. pmid=TBA [TBA]
  6. Sinnott ML and Souchard IJ. The mechanism of action of beta-galactosidase. Effect of aglycone nature and -deuterium substitution on the hydrolysis of aryl galactosides. Biochem J. 1973 May;133(1):89-98. PubMed ID:4578762 | HubMed [SinnottSouchard1973]
  7. Sinnott ML and Viratelle OM. 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 May;133(1):81-7. PubMed ID:4721624 | HubMed [SinnottViratelle1973]
  8. Viratelle OM and Yon JM. Nucleophilic competition in some -galactosidase-catalyzed reactions. Eur J Biochem. 1973 Feb 15;33(1):110-6. PubMed ID:4691347 | HubMed [ViratelleYon1973]
  9. McCarter JD, Adam MJ, and Withers SG. Binding energy and catalysis. Fluorinated and deoxygenated glycosides as mechanistic probes of Escherichia coli (lacZ) beta-galactosidase. Biochem J. 1992 Sep 15;286 ( Pt 3):721-7. PubMed ID:1417731 | HubMed [McCarter1992]
  10. Gebler JC, Aebersold R, and Withers SG. Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) beta-galactosidase from Escherichia coli. J Biol Chem. 1992 Jun 5;267(16):11126-30. PubMed ID:1350782 | HubMed [Gebler1992]
  11. Herrchen M and Legler G. Identification of an essential carboxylate group at the active site of lacZ beta-galactosidase from Escherichia coli. Eur J Biochem. 1984 Feb 1;138(3):527-31. PubMed ID:6420154 | HubMed [HerrchenLegler1984]
  12. Cupples CG, Miller JH, and Huber RE. Determination of the roles of Glu-461 in beta-galactosidase (Escherichia coli) using site-specific mutagenesis. J Biol Chem. 1990 Apr 5;265(10):5512-8. PubMed ID:1969405 | HubMed [Cupples1990]
  13. Jacobson RH, Zhang XJ, DuBose RF, and Matthews BW. Three-dimensional structure of beta-galactosidase from E. coli. Nature. 1994 Jun 30;369(6483):761-6. DOI:10.1038/369761a0 | PubMed ID:8008071 | HubMed [Jacobson1994]
All Medline abstracts: PubMed | HubMed
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