Lactase

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Lactase
Identifiers
EC no.3.2.1.108
CAS no.9031-11-2
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / QuickGO
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PMCarticles
PubMedarticles
NCBIproteins
Glycosylceramidase
(Phlorizin hydrolase)
Identifiers
EC no.3.2.1.62
CAS no.9033-10-7
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / QuickGO
Search
PMCarticles
PubMedarticles
NCBIproteins
Lactase
Identifiers
SymbolLCT
Alt. symbolsLAC; LPH; LPH1
NCBI gene3938
HGNC6530
OMIM603202
RefSeqNM_002299
UniProtP09848
Other data
EC number3.2.1.108
LocusChr. 2 q21
Search for
StructuresSwiss-model
DomainsInterPro

Lactase (also known as lactase-phlorizin hydrolase, or LPH), a part of the β-galactosidase family of enzymes, is a glycoside hydrolase involved in the hydrolysis of the disaccharide lactose into constituent galactose and glucose monomers. Lactase is present predominantly along the brush border membrane of the differentiated enterocytes lining the villi of the small intestine.[1] In humans, lactase is encoded by the LCT gene.[2][3]

Lactase is essential for digestive hydrolysis of lactose in milk. Deficiency of the enzyme causes lactose intolerance.[4]

The optimum temperature for lactase is about 77 °F (25 °C) for its activity[5] and has an optimum pH of 6.[1]

Function and mechanism

Lactase can hydrolyze a variety of substrates. While it is most notably a member of the β-galactosidase enzymatic class, lactase also has glucosidase and glycosylceramidase activity.[6] In metabolism, the β-glycosidic bond in D-lactose is hydrolyzed to form D-galactose and D-glucose, which can be absorbed through the intestinal walls and into the bloodstream. The overall reaction that lactase catalyzes is C12H22O11 + H2O → C6H12O6 + C6H12O6 + heat. Lactase also catalyzes the conversion of phlorizin to phloretin and glucose.

The catalytic mechanism of D-lactose hydrolysis retains the substrate anomeric configuration in the products.[7] While the details of the mechanism are uncertain, the stereochemical retention is achieved through a double displacement reaction. Studies of E. coli lactase have proposed that hydrolysis is initiated when a glutamate nucleophile on the enzyme attacks from the axial side of the galactosyl carbon in the β-glycosidic bond.[8] The removal of the D-glucose leaving group may be facilitated by Mg-dependent acid catalysis.[8] The enzyme is liberated from the α-galactosyl moiety upon equatorial nucleophilic attack by water, which produces D-galactose.[7]

Substrate modification studies have demonstrated that the 3’-OH and 2’-OH moieties on the galactopyranose ring are essential for enzymatic recognition and hydrolysis.[6] The 3’-hydroxy group is involved in initial binding to the substrate while the 2’- group is not necessary for recognition but needed in subsequent steps. This is demonstrated by the fact that a 2-deoxy analog is an effective competitive inhibitor (Ki = 10mM).[6] Elimination of specific hydroxyl groups on the glucopyranose moiety does not completely eliminate catalysis.[6]

Proposed mechanism of lactose hydrolysis.

Structure and biosynthesis

Pre-pro-lactase, the primary translation product, has a single polypeptide primary structure consisting of 1927 amino acids.[9] It can be divided into five domains: (i) a 19 amino acid cleaved signal sequence; (ii) a large prosequence domain that is not present in mature lactase; (iii) the mature lactase segment; (iv) a membrane spanning hydrophobic anchor; and (v) a short hydrophilic carboxyl terminus.[2] The signal sequence is cleaved in the endoplasmic reticulum, and the resulting 215 kDa pro-LPH is sent to the Golgi, where it is heavily glycosylated and proteolytically processed to its mature form.[10] The prodomain has been shown to act as an intramolecular chaperone in the ER, preventing trypsin cleavage and allowing LPH to adopt the necessary 3-D structure to be transported to the Golgi apparatus.[11]

Schematic of processing and localization of human lactase translational product.

Mature human lactase consists of a single 160 kDa polypeptide chain that localizes to the brush border membrane of intestinal epithelial cells. It is oriented with the N-terminus outside the cell and the C-terminus in the cytosol.[2] LPH contains two catalytic glutamic acid sites. In the human enzyme, the lactase activity has been connected to Glu-1749 while Glu-1273 is the site of phlorizin hydrolase function.[12]

Genetic expression and regulation

Lactase is encoded by a single genetic locus on chromosome 2.[13] It is expressed exclusively by mammalian small intestine enterocytes and in very low levels in the colon during fetal development.[13] Humans are born with high levels of lactase expression. In most of the world’s population, lactase transcription is down-regulated after weaning, resulting in diminished lactase expression in the small intestine.[13] Diminished lactase expression causes the common symptoms of adult-type hypolactasia, or lactose intolerance.

Some population segments exhibit lactase persistence resulting from a mutation that is postulated to have occurred 5000-10,000 years ago, coinciding with the rise of cattle domestication.[14] This mutation has allowed almost half of the world’s population to metabolize lactose without symptoms. Studies have linked the occurrence of lactase persistence to two different single-nucleotide polymorphisms about 14 and 22 kilobases upstream of the 5’-end of the LPH gene.[15] Both mutations, C→T at position -13910 and T→ A at position -22018, have been independently linked to lactase persistence.[16]

The lactase promoter is 150 base pairs long and is located just upstream of the site of transcription initiation.[16] The sequence is highly conserved in mammals, suggesting that critical cis-transcriptional regulators are located nearby.[16] Cdx-2, HNF-1α, and GATA have been identified as transcription factors.[16] Studies of hypolactasia onset have demonstrated that despite polymorphisms there is little difference in lactase expression in infants, showing that the mutations become increasingly relevant during development.[17] Ιt is hypothesized that developmentally-regulated DNA-binding proteins down-regulate transcription or destabilize mRNA transcripts, causing decreased LPH expression after weaning.[17]

Industrial use

Lactase produced commercially can be extracted both from yeasts such as Kluyveromyces fragilis and Kluyveromyces lactis and from fungi, such as Aspergillus niger and Aspergillus oryzae.[18] Its primary commercial use is to break down lactose in milk to make it suitable for people with lactose intolerance. However, the U.S. Food and Drug Administration (FDA) has not formally evaluated the effectiveness of these products.[19] ].

Lactase is also used to screen for blue white colonies in the MCS of various plasmid vectors in Escherichia coli or other bacteria.[citation needed]

See also

References

  1. ^ a b Skovbjerg H, Sjöström H, Norén O (1981). "Purification and characterisation of amphiphilic lactase/phlorizin hydrolase from human small intestine". Eur. J. Biochem. 114 (3): 653–61. doi:10.1111/j.1432-1033.1981.tb05193.x. PMID 6786877. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  2. ^ a b c Mantei N, Villa M, Enzler T, Wacker H, Boll W, James P, Hunziker W, Semenza G (1988). "Complete primary structure of human and rabbit lactase-phlorizin hydrolase: implications for biosynthesis, membrane anchoring and evolution of the enzyme". EMBO J. 7 (9): 2705–13. PMC 457059. PMID 2460343. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  3. ^ Harvey CB, Fox MF, Jeggo PA, Mantei N, Povey S, Swallow DM (1993). "Regional localization of the lactase-phlorizin hydrolase gene, LCT, to chromosome 2q21". Ann. Hum. Genet. 57 (Pt 3): 179–85. doi:10.1111/j.1469-1809.1993.tb01593.x. PMID 8257087. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  4. ^ Järvelä I, Torniainen S, Kolho KL (2009). "Molecular genetics of human lactase deficiencies". Ann. Med. 41 (8): 568–75. doi:10.1080/07853890903121033. PMID 19639477.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  5. ^ Hermida C, Corrales G, Cañada FJ, Aragón JJ, Fernández-Mayoralas A (2007). "Optimizing the enzymatic synthesis of beta-D-galactopyranosyl-D-xyloses for their use in the evaluation of lactase activity in vivo". Bioorg. Med. Chem. 15 (14): 4836–40. doi:10.1016/j.bmc.2007.04.067. PMID 17512743. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  6. ^ a b c d Fernandez P, Cañada F, Jiménez-Barbero J, Martín-Lomas, M (1995). "Substrate specificity of small-intestinal lactase: Study of the steric effects and hydrogen bonds involved in enzyme-substrate interaction". Carbohydr. Res. 271 (1): 31–42. doi:10.1016/0008-6215(95)00034-Q. PMID 7648581. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  7. ^ a b Sinnott M (1990). "Catalytic mechanisms of enzymic glycosyl transfer". Chem. Rev. 90 (7): 1171–1202. doi:10.1021/cr00105a006. {{cite journal}}: Unknown parameter |month= ignored (help)
  8. ^ a b Juers D, Heightman T, Vasella A, McCarter J, Mackenzie L, Withers S, Matthews B (2001). "A structural view of the action of Escherichia coli (lacZ) β-galactosidase". Biochemistry. 40 (49): 14781–94. doi:10.1021/bi011727i. PMID 11732897. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  9. ^ Cite error: The named reference ref name= was invoked but never defined (see the help page).
  10. ^ Naim HY, Sterchi E, Lentze M (1987). "Biosynthesis and maturation of lactase-phlorizin hydrolase in the human small intestinal epithelial cells". Biochem. J. 241 (2): 427–34. PMC 1147578. PMID 3109375. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  11. ^ Naim HY, Jacob R, Naim H, Sambrook J, Gething M (1994). "The pro region of human intestinal lactase-phlorizin hydrolase". J. Biol. Chem. 269 (43): 26933–43. PMID 7523415. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  12. ^ Zecca L, Mesonero J, Stutz A, Poirée J, Guidicelli J, Cursio R, Gloor S, Semenza G (1998). "Intestinal lactase-phlorizin hydrolase (LPH): the two catalytic sites; the role of the pancreas in pro-LPH maturation". FEBS Lett. 435 (2–3): 225–8. doi:10.1016/S0014-5793(98)01076-X. PMID 9762914. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  13. ^ a b c Troelsen J, Mitchelmore C, Spodsberg N, Jensen A, Norén O, Sjöström H (1997). "Regulation of lactase-phlorizin hydrolase gene expression by the caudal-related homoeodomain protein Cdx-2". Biochem. J. 322 (Pt. 3): 833–838. PMC 1218263. PMID 9148757. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  14. ^ Bersaglier T, Sabeti P, Patterson N, Vanderploeg T, Schaffner S, Drake J, Rhodes M, Reich D, Hirschhorn J (2004). "Genetic Signatures of Strong Recent Positive Selection at the Lactase Gene". Am. J. Hum. Gen. 74 (6): 1111–20. doi:10.1086/421051. PMC 1182075. PMID 15114531. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  15. ^ Kuokkanen M, Enattah N, Oksanen A, Savilhahtl E, Orpana A, Järvela I (2003). "Transcriptional regulation of the lactase-phlorizin hydrolase gene by polymorphisms associated with adult-type hypolactasia". Gut. 52 (5): 647–52. doi:10.1136/gut.52.5.647. PMC 1773659. PMID 12692047. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  16. ^ a b c d Troelsen J (2005). "Adult-type hypolactasia and regulation of lactase expression". Biochim Biophys Acta. 1723 (1–3): 19–32. doi:10.1016/j.bbagen.2005.02.003. PMID 15777735. {{cite journal}}: Unknown parameter |month= ignored (help)
  17. ^ a b Wang Y, Harvey C, Hollox E, Phillips A, Poulter M, Clay P, Walker-Smith J, Swallow D (1998). "The genetically programmed down-regulation of lactase in children". Gastroenterology. 114 (6): 1230–6. doi:10.1016/S0016-5085(98)70429-9. PMID 9609760. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  18. ^ Seyis I, Aksoz N (2004). "Production of lactase by Trichoderma sp" (PDF). Food Technol Biotechnol. 42: 121–124.
  19. ^ Tarantino, LM (2003-12-03). "Agency Response Letter GRAS Notice No. GRN 000132". U.S. Food and Drug Administration. Retrieved 2009-09-21.

External links

  1. ^ Beebe, Jane A.; Frey, Perry A. (1998-10-01). "Galactose Mutarotase: Purification, Characterization, and Investigations of Two Important Histidine Residues". Biochemistry. 37 (42): 14989–14997. doi:10.1021/bi9816047. ISSN 0006-2960.
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