Lactase

Lactase

Lactase tetramer, E.Coli
Identifiers
EC number 3.2.1.108
CAS number 9031-11-2
Databases
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Gene Ontology AmiGO / EGO
Glycosylceramidase
(Phlorizin hydrolase)
Identifiers
EC number 3.2.1.62
CAS number 9033-10-7
Databases
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Gene Ontology AmiGO / EGO
Lactase
Identifiers
Symbol LCT
Alt. symbols LAC; LPH; LPH1
Entrez 3938
HUGO 6530
OMIM 603202
RefSeq NM_002299
UniProt P09848
Other data
EC number 3.2.1.108
Locus Chr. 2 q21

Lactase is an enzyme produced by many organisms. It is located in the brush border of the small intestine of humans and other mammals. Lactase is essential to the complete digestion of whole milk; it breaks down lactose, a sugar which gives milk its sweetness. Lacking lactase, a person consuming dairy products may experience the symptoms of lactose intolerance.[1] Lactase can be purchased as a food supplement, and is added to milk to produce "lactose-free" milk products.

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.[2] In humans, lactase is encoded by the LCT gene.[3][4]

Uses

Medical use

Lactase supplements are sometimes used to treat lactose intolerance.[5]

Industrial use

Lactase produced commercially can be extracted both from yeasts such as Kluyveromyces fragilis and Kluyveromyces lactis and from molds, such as Aspergillus niger and Aspergillus oryzae.[6] Its primary commercial use, in supplements such as Lacteeze and Lactaid, is to break down lactose in milk to make it suitable for people with lactose intolerance,[7][8] However, the U.S. Food and Drug Administration has not formally evaluated the effectiveness of these products.[9]

Lactase is also used to screen for blue white colonies in the multiple cloning sites of various plasmid vectors in Escherichia coli or other bacteria.

Mechanism

The optimum temperature for human lactase is about 37 °C for its activity[10] and has an optimum pH of 6.[2]

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.

The catalytic mechanism of D-lactose hydrolysis retains the substrate anomeric configuration in the products.[11] 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.[12] The removal of the D-glucose leaving group may be facilitated by Mg-dependent acid catalysis.[12] The enzyme is liberated from the α-galactosyl moiety upon equatorial nucleophilic attack by water, which produces D-galactose.[11]

Substrate modification studies have demonstrated that the 3′-OH and 2′-OH moieties on the galactopyranose ring are essential for enzymatic recognition and hydrolysis.[13] 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).[13] Elimination of specific hydroxyl groups on the glucopyranose moiety does not completely eliminate catalysis.[13]

Lactase also catalyzes the conversion of phlorizin to phloretin and glucose.

Structure and biosynthesis

Preprolactase, the primary translation product, has a single polypeptide primary structure consisting of 1927 amino acids.[3] 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.[3] The signal sequence is cleaved in the endoplasmic reticulum, and the resulting 215-kDa pro-LPH is sent to the Golgi apparatus, where it is heavily glycosylated and proteolytically processed to its mature form.[14] 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.[15]

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.[3] 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.[16]

Genetic expression and regulation

Lactase is encoded by a single genetic locus on chromosome 2.[17] It is expressed exclusively by mammalian small intestine enterocytes and in very low levels in the colon during fetal development.[17] 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,[17] which causes the common symptoms of adult-type hypolactasia, or lactose intolerance.[18]

Some population segments exhibit lactase persistence resulting from a mutation that is postulated to have occurred 5,000-10,000 years ago, coinciding with the rise of cattle domestication.[19] 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.[20] Both mutations, C→T at position -13910 and G→ A at position -22018, have been independently linked to lactase persistence.[21]

The lactase promoter is 150 base pairs long and is located just upstream of the site of transcription initiation.[21] The sequence is highly conserved in mammals, suggesting that critical cis-transcriptional regulators are located nearby.[21] Cdx-2, HNF-1α, and GATA have been identified as transcription factors.[21] Studies of hypolactasia onset have demonstrated that despite polymorphisms, little difference exists in lactase expression in infants, showing that the mutations become increasingly relevant during development.[22] Developmentally regulated DNA-binding proteins may down-regulate transcription or destabilize mRNA transcripts, causing decreased LPH expression after weaning.[22]

See also

References

  1. ↑ Järvelä I, Torniainen S, Kolho KL (2009). "Molecular genetics of human lactase deficiencies". Annals of Medicine. 41 (8): 568–75. doi:10.1080/07853890903121033. PMID 19639477.
  2. 1 2 Skovbjerg H, Sjöström H, Norén O (Mar 1981). "Purification and characterisation of amphiphilic lactase/phlorizin hydrolase from human small intestine". European Journal of Biochemistry / FEBS. 114 (3): 653–61. doi:10.1111/j.1432-1033.1981.tb05193.x. PMID 6786877.
  3. 1 2 3 4 Mantei N, Villa M, Enzler T, Wacker H, Boll W, James P, Hunziker W, Semenza G (Sep 1988). "Complete primary structure of human and rabbit lactase-phlorizin hydrolase: implications for biosynthesis, membrane anchoring and evolution of the enzyme". The EMBO Journal. 7 (9): 2705–13. PMC 457059Freely accessible. PMID 2460343.
  4. ↑ Harvey CB, Fox MF, Jeggo PA, Mantei N, Povey S, Swallow DM (Jul 1993). "Regional localization of the lactase-phlorizin hydrolase gene, LCT, to chromosome 2q21". Annals of Human Genetics. 57 (Pt 3): 179–85. doi:10.1111/j.1469-1809.1993.tb01593.x. PMID 8257087.
  5. ↑ "Lactose Intolerance". NIDDK. June 2014. Retrieved 25 October 2016.
  6. ↑ Seyis I, Aksoz N (2004). "Production of lactase by Trichoderma sp" (PDF). Food Technol Biotechnol. 42: 121–124.
  7. ↑ "Re: GRAS Notification for Acid Lactase from Aspergillus oryzae Expressed in Aspergillus niger". United States Food and Drug Administration.
  8. ↑ Holsinger VH. "The Lactaid Story". Innovative Products for Food Industries. pp. 256–8.
  9. ↑ Tarantino, LM (2003-12-03). "Agency Response Letter GRAS Notice No. GRN 000132". U.S. Food and Drug Administration. Retrieved 2009-09-21.
  10. ↑ Hermida C, Corrales G, Cañada FJ, Aragón JJ, Fernández-Mayoralas A (Jul 2007). "Optimizing the enzymatic synthesis of beta-D-galactopyranosyl-D-xyloses for their use in the evaluation of lactase activity in vivo". Bioorganic & Medicinal Chemistry. 15 (14): 4836–40. doi:10.1016/j.bmc.2007.04.067. PMID 17512743.
  11. 1 2 Sinnott M (November 1990). "Catalytic mechanisms of enzymic glycosyl transfer". Chem. Rev. 90 (7): 1171–1202. doi:10.1021/cr00105a006.
  12. 1 2 Juers DH, Heightman TD, Vasella A, McCarter JD, Mackenzie L, Withers SG, Matthews BW (Dec 2001). "A structural view of the action of Escherichia coli (lacZ) beta-galactosidase". Biochemistry. 40 (49): 14781–94. doi:10.1021/bi011727i. PMID 11732897.
  13. 1 2 3 Fernandez P, Cañada FJ, Jiménez-Barbero J, Martín-Lomas M (Jul 1995). "Substrate specificity of small-intestinal lactase: study of the steric effects and hydrogen bonds involved in enzyme-substrate interaction". Carbohydrate Research. 271 (1): 31–42. doi:10.1016/0008-6215(95)00034-Q. PMID 7648581.
  14. ↑ Naim HY, Sterchi EE, Lentze MJ (Jan 1987). "Biosynthesis and maturation of lactase-phlorizin hydrolase in the human small intestinal epithelial cells". The Biochemical Journal. 241 (2): 427–34. PMC 1147578Freely accessible. PMID 3109375.
  15. ↑ Naim HY, Jacob R, Naim H, Sambrook JF, Gething MJ (Oct 1994). "The pro region of human intestinal lactase-phlorizin hydrolase". The Journal of Biological Chemistry. 269 (43): 26933–43. PMID 7523415.
  16. ↑ Zecca L, Mesonero JE, Stutz A, Poirée JC, Giudicelli J, Cursio R, Gloor SM, Semenza G (Sep 1998). "Intestinal lactase-phlorizin hydrolase (LPH): the two catalytic sites; the role of the pancreas in pro-LPH maturation". FEBS Letters. 435 (2-3): 225–8. doi:10.1016/S0014-5793(98)01076-X. PMID 9762914.
  17. 1 2 3 Troelsen JT, Mitchelmore C, Spodsberg N, Jensen AM, Norén O, Sjöström H (Mar 1997). "Regulation of lactase-phlorizin hydrolase gene expression by the caudal-related homoeodomain protein Cdx-2". The Biochemical Journal. 322 ( Pt 3) (Pt. 3): 833–8. PMC 1218263Freely accessible. PMID 9148757.
  18. ↑ https://ghr.nlm.nih.gov/gene/LCT
  19. ↑ Bersaglieri T, Sabeti PC, Patterson N, Vanderploeg T, Schaffner SF, Drake JA, Rhodes M, Reich DE, Hirschhorn JN (Jun 2004). "Genetic signatures of strong recent positive selection at the lactase gene". American Journal of Human Genetics. 74 (6): 1111–20. doi:10.1086/421051. PMC 1182075Freely accessible. PMID 15114531.
  20. ↑ Kuokkanen M, Enattah NS, Oksanen A, Savilahti E, Orpana A, Järvelä I (May 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 1773659Freely accessible. PMID 12692047.
  21. 1 2 3 4 Troelsen JT (May 2005). "Adult-type hypolactasia and regulation of lactase expression". Biochimica et Biophysica Acta. 1723 (1-3): 19–32. doi:10.1016/j.bbagen.2005.02.003. PMID 15777735.
  22. 1 2 Wang Y, Harvey CB, Hollox EJ, Phillips AD, Poulter M, Clay P, Walker-Smith JA, Swallow DM (Jun 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.
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