Lactase

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Lactase
1jyn.jpg
E. coli lactase tetramer. PDB: 1JYN ​
Identifiers
EC no. 3.2.1.108
CAS no. 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 / QuickGO
Search
PMC articles
PubMed articles
NCBI proteins
Lactase
Identifiers
SymbolLCT
Alt. symbolsLAC; LPH; LPH1
NCBI gene 3938
HGNC 6530
OMIM 603202
RefSeq NM_002299
UniProt P09848
Other data
EC number 3.2.1.108
Locus Chr. 2 q21
Search for
Structures Swiss-model
Domains InterPro

Lactase (EC 3.2.1.108) is an enzyme produced by many organisms and is essential to the complete digestion of whole milk. It breaks down the sugar lactose into its component parts, galactose and glucose. Lactase is found in the brush border of the small intestine of humans and other mammals. People deficient in lactase or lacking functional lactase may experience the symptoms of lactose intolerance after consuming milk products. [1] Lactase can be purchased as a food supplement and is added to milk to produce "lactose-free" milk products.

Contents

Uses

Food use

Lactase is an enzyme that some people are unable to produce in their small intestine. [2] Technology to produce lactose-free milk, ice cream, and yogurt was developed by the USDA Agricultural Research Service in 1985. [3] This technology is used to add lactase to milk, thereby hydrolyzing the lactose naturally found in milk, leaving it slightly sweet but digestible by everyone. [4] Without lactase, lactose intolerant people pass the lactose undigested to the colon [5] where bacteria break it down, creating carbon dioxide which leads to bloating and flatulence.

Medical use

Lactase supplements can be used to treat lactose intolerance. [6]

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 . [7] Its primary commercial use in supplements is to break down lactose in milk to make it suitable for people with lactose intolerance. [8] [9] The U.S. Food and Drug Administration has not independently evaluated these products. [10]

Lactase (or a similar form of β-galactosidase) is also used to screen for blue white colonies in the multiple cloning sites of various plasmid vectors in Escherichia coli or other bacteria. [11] [12]

Mechanism

The optimum temperature for human lactase is about 37 °C [13] and the optimum pH is 6. [14]

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 as follows:

C12H22O11 + H2O → C6H12O6 + C6H12O6 + heat.
lactose + H2O → β-D-galactose + D-glucose

The catalytic mechanism of D-lactose hydrolysis retains the substrate anomeric configuration in the products. [15] While the details of the mechanism are uncertain, the stereochemical retention is achieved off 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. [16] The removal of the D-glucose leaving group may be facilitated by Mg-dependent acid catalysis. [16] The enzyme is liberated from the α-galactosyl moiety upon equatorial nucleophilic attack by water, which produces D-galactose. [15]

Substrate modification studies have demonstrated that the 3′-OH and 2′-OH moieties on the galactopyranose ring are essential for enzymatic recognition and hydrolysis. [17] 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). [17] Elimination of specific hydroxyl groups on the glucopyranose moiety does not eliminate catalysis. [17]

LactaseMechanism2.png

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

Lactase (Lactaid commercially) is used as a medication for lactose intolerance. Since it is an enzyme, its function can be inhibited by the acidity of the stomach. However, it is packaged in an acid-proof tablet, allowing the enzyme to pass through the stomach intact and remain in the small intestine. In the small intestine it can act on ingested lactose molecules, allowing the body to absorb the digested sugar which would otherwise cause cramping and diarrhea. Since the enzyme is not absorbed, it is excreted during the next bowel movement.[ citation needed ]

Structure and biosynthesis

Preprolactase, the primary translation product, has a single polypeptide primary structure consisting of 1927 amino acids. [18] 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. [18] 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. [19] 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. [20]

Lactase Processing.png

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. [18] 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. [21]

Genetic expression and regulation

Lactase is encoded by a single genetic locus on chromosome 2. [22] It is expressed exclusively by mammalian small intestine enterocytes and in very low levels in the colon during fetal development. [22] 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, [22] which causes the common symptoms of adult-type hypolactasia, or lactose intolerance. [23] The LCT gene provides the instructions for making lactase. Lactose intolerance in infants (congenital lactase deficiency) is caused by mutations in the LCT gene. Mutations are believed to interfere with the function of lactase, causing affected infants to have a severely impaired ability to digest lactose in breast milk or formula. [24]

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. [25] 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. [26] Both mutations, C→T at position -13910 and G→ A at position -22018, have been independently linked to lactase persistence. [27]

The lactase promoter is 150 base pairs long and is located upstream of the site of transcription initiation. [27] The sequence is highly conserved in mammals, suggesting that critical cis-transcriptional regulators are located nearby. [27] Cdx-2, HNF-1α, and GATA have been identified as transcription factors. [27] 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. [28] Developmentally regulated DNA-binding proteins may down-regulate transcription or destabilize mRNA transcripts, causing decreased LPH expression after weaning. [28]

See also

Related Research Articles

<span class="mw-page-title-main">Lactose</span> Carbohydrate

Lactose, or milk sugar, is a disaccharide sugar composed of galactose and glucose subunits and has the molecular formula C12H22O11. Lactose makes up around 2–8% of milk (by mass). The name comes from lact (gen. lactis), the Latin word for milk, plus the suffix -ose used to name sugars. The compound is a white, water-soluble, non-hygroscopic solid with a mildly sweet taste. It is used in the food industry.

<span class="mw-page-title-main">Galactose</span> Monosaccharide sugar

Galactose, sometimes abbreviated Gal, is a monosaccharide sugar that is about as sweet as glucose, and about 65% as sweet as sucrose. It is an aldohexose and a C-4 epimer of glucose. A galactose molecule linked with a glucose molecule forms a lactose molecule.

<span class="mw-page-title-main">Lactose intolerance</span> Inability to digest lactose

Lactose intolerance is caused by a lessened ability or a complete inability to digest lactose, a sugar found in dairy products. Humans vary in the amount of lactose they can tolerate before symptoms develop. Symptoms may include abdominal pain, bloating, diarrhea, flatulence, and nausea. These symptoms typically start thirty minutes to two hours after eating or drinking something containing lactose, with the severity typically depending on the amount consumed. Lactose intolerance does not cause damage to the gastrointestinal tract.

β-Galactosidase Family of glycoside hydrolase enzymes

β-Galactosidase is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides.

<span class="mw-page-title-main">François Jacob</span> French biologist

François Jacob was a French biologist who, together with Jacques Monod, originated the idea that control of enzyme levels in all cells occurs through regulation of transcription. He shared the 1965 Nobel Prize in Medicine with Jacques Monod and André Lwoff.

<i>lac</i> operon Set genes encoding proteins and enzymes for lactose metabolism

The lactose operon is an operon required for the transport and metabolism of lactose in E. coli and many other enteric bacteria. Although glucose is the preferred carbon source for most enteric bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available through the activity of beta-galactosidase. Gene regulation of the lac operon was the first genetic regulatory mechanism to be understood clearly, so it has become a foremost example of prokaryotic gene regulation. It is often discussed in introductory molecular and cellular biology classes for this reason. This lactose metabolism system was used by François Jacob and Jacques Monod to determine how a biological cell knows which enzyme to synthesize. Their work on the lac operon won them the Nobel Prize in Physiology in 1965.

<span class="mw-page-title-main">Galactosemia</span> Medical condition

Galactosemia is a rare genetic metabolic disorder that affects an individual's ability to metabolize the sugar galactose properly. Galactosemia follows an autosomal recessive mode of inheritance that confers a deficiency in an enzyme responsible for adequate galactose degradation.

<span class="mw-page-title-main">Enterocyte</span> Type of intestinal cell

Enterocytes, or intestinal absorptive cells, are simple columnar epithelial cells which line the inner surface of the small and large intestines. A glycocalyx surface coat contains digestive enzymes. Microvilli on the apical surface increase its surface area. This facilitates transport of numerous small molecules into the enterocyte from the intestinal lumen. These include broken down proteins, fats, and sugars, as well as water, electrolytes, vitamins, and bile salts. Enterocytes also have an endocrine role, secreting hormones such as leptin.

Lactase persistence is the continued activity of the lactase enzyme in adulthood, allowing the digestion of lactose in milk. In most mammals, the activity of the enzyme is dramatically reduced after weaning. In some human populations though, lactase persistence has recently evolved as an adaptation to the consumption of nonhuman milk and dairy products beyond infancy. Lactase persistence is very high among northern Europeans, especially Irish people. Worldwide, most people are lactase non-persistent, and are affected by varying degrees of lactose intolerance as adults. However, lactase persistence and lactose intolerance can overlap.

Disaccharidases are glycoside hydrolases, enzymes that break down certain types of sugars called disaccharides into simpler sugars called monosaccharides. In the human body, disaccharidases are made mostly in an area of the small intestine's wall called the brush border, making them members of the group of "brush border enzymes".

α-Galactosidase Enzyme

α-Galactosidase is a glycoside hydrolase enzyme that catalyses the following reaction:

Galactosidases are enzymes that catalyze the hydrolysis of galactosides into monosaccharides.

<span class="mw-page-title-main">Glycoside hydrolase</span> Class of enzymes which break glycosidic bonds via hydrolysis

In biochemistry, glycoside hydrolases are a class of enzymes which catalyze the hydrolysis of glycosidic bonds in complex sugars. They are extremely common enzymes, with roles in nature including degradation of biomass such as cellulose (cellulase), hemicellulose, and starch (amylase), in anti-bacterial defense strategies, in pathogenesis mechanisms and in normal cellular function. Together with glycosyltransferases, glycosidases form the major catalytic machinery for the synthesis and breakage of glycosidic bonds.

<span class="mw-page-title-main">Galactose-1-phosphate uridylyltransferase deficiency</span> Medical condition

Galactose-1-phosphate uridylyltransferase deficiency(classic galactosemia) is the most common type of galactosemia, an inborn error of galactose metabolism, caused by a deficiency of the enzyme galactose-1-phosphate uridylyltransferase. It is an autosomal recessive metabolic disorder that can cause liver disease and death if untreated. Treatment of galactosemia is most successful if initiated early and includes dietary restriction of lactose intake. Because early intervention is key, galactosemia is included in newborn screening programs in many areas. On initial screening, which often involves measuring the concentration of galactose in blood, classic galactosemia may be indistinguishable from other inborn errors of galactose metabolism, including galactokinase deficiency and galactose epimerase deficiency. Further analysis of metabolites and enzyme activities are needed to identify the specific metabolic error.

<span class="mw-page-title-main">Glucose-galactose malabsorption</span> Medical condition

Glucose-galactose malabsorption is a rare condition in which the cells lining the intestine cannot take in the sugars glucose and galactose, which prevents proper digestion of these molecules and larger molecules made from them.

The enzyme glycosylceramidase (EC 3.2.1.62) catalyzes the following chemical reaction:

<span class="mw-page-title-main">Inborn errors of carbohydrate metabolism</span> Medical condition

Inborn errors of carbohydrate metabolism are inborn error of metabolism that affect the catabolism and anabolism of carbohydrates.

Carbon catabolite repression, or simply catabolite repression, is an important part of global control system of various bacteria and other microorganisms. Catabolite repression allows microorganisms to adapt quickly to a preferred carbon and energy source first. This is usually achieved through inhibition of synthesis of enzymes involved in catabolism of carbon sources other than the preferred one. The catabolite repression was first shown to be initiated by glucose and therefore sometimes referred to as the glucose effect. However, the term "glucose effect" is actually a misnomer since other carbon sources are known to induce catabolite repression.

<span class="mw-page-title-main">Glycoside hydrolase family 27</span>

In molecular biology, glycoside hydrolase family 27 is a family of glycoside hydrolases.

Lacto-<i>N</i>-tetraose Chemical compound

Lacto-N-tetraose is a complex sugar found in human milk. It is one of the few characterized human milk oligosaccharides (HMOs) and is enzymatically synthesized from the substrate lactose. It is biologically relevant in the early development of the infant gut flora.

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. S2CID   205586720.
  2. ↑ "Lactose Intolerance". Mayo Clinic. Retrieved 13 March 2018.
  3. ↑ Porch K (2018-04-12). "Lactose-Free Milk, Low-Fat Cheese, and More Dairy Breakthroughs". www.federallabs.org. Retrieved 2018-10-26.
  4. ↑ "Asked: How do dairies make lactose-free milk?". USA Today. 3 September 2014. Retrieved 13 March 2018.
  5. ↑ "Lactose intolerance - Symptoms and causes". Mayo Clinic. Retrieved 2020-11-08.
  6. ↑ "Lactose Intolerance". NIDDK. June 2014. Retrieved 25 October 2016.
  7. ↑ Seyis I, Aksoz N (2004). "Production of Lactase by Trichoderma sp". Food Technology and Biotechnology. 42 (2): 121–124.
  8. ↑ DSM Food Specialties (3 April 2014). "GRAS Notification for Acid Lactase from Aspergillus oryzae Expressed in Aspergillus niger" (PDF). p. 1. Archived from the original on 31 October 2017 – via U.S. Food and Drug Administration.
  9. ↑ Holsinger VH (1992). "Innovative Products for Food Industries: The Lactaid Story". New Crops, New Uses, New Markets: 1992 Yearbook of Agriculture. U.S. Department of Agriculture. pp. 256–258. Archived from the original on 2022-07-17. Retrieved 2022-01-11.
  10. ↑ Tarantino, LM (12 December 2003). "Agency Response Letter GRAS Notice No. GRN 000132". U.S. Food and Drug Administration. Archived from the original on 26 March 2011.
  11. ↑ Lau HM, Lee LS, Soh WC, Tue SW (March 2013). "Introduction". Lactase. Universiti Teknologi Malaysia. Retrieved 16 November 2018.
  12. ↑ "pBluescript II KS(+/−), pBluescript II SK(+/−): description & restriction map". Fermentas. Archived from the original on 19 October 2008.
  13. ↑ Hermida C, Corrales G, Cañada FJ, Aragón JJ, Fernández-Mayoralas A (Jul 2007). "Optimizing the enzymatic synthesis of β-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. hdl:10261/81580. PMID   17512743.
  14. ↑ Skovbjerg H, Sjöström H, Norén O (Mar 1981). "Purification and characterisation of amphiphilic lactase/phlorizin hydrolase from human small intestine". European Journal of Biochemistry. 114 (3): 653–61. doi:10.1111/j.1432-1033.1981.tb05193.x. PMID   6786877.
  15. 1 2 Sinnott M (November 1990). "Catalytic mechanisms of enzymic glycosyl transfer". Chem. Rev. 90 (7): 1171–1202. doi:10.1021/cr00105a006.
  16. 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) β-galactosidase". Biochemistry. 40 (49): 14781–94. doi:10.1021/bi011727i. PMID   11732897.
  17. 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.
  18. 1 2 3 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. doi:10.1002/j.1460-2075.1988.tb03124.x. PMC   457059 . PMID   2460343.
  19. ↑ 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. doi:10.1042/bj2410427. PMC   1147578 . PMID   3109375.
  20. ↑ 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. doi: 10.1016/S0021-9258(18)47109-8 . PMID   7523415.
  21. ↑ 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. S2CID   33421778.
  22. 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. doi:10.1042/bj3220833. PMC   1218263 . PMID   9148757.
  23. ↑ Reference GH. "LCT gene". Genetics Home Reference. Retrieved 3 April 2018.
  24. ↑ "Lactose intolerance: MedlinePlus Genetics". medlineplus.gov. Retrieved 2022-03-22.
  25. ↑ 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   1182075 . PMID   15114531.
  26. ↑ 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   1773659 . PMID   12692047.
  27. 1 2 3 4 Troelsen JT (May 2005). "Adult-type hypolactasia and regulation of lactase expression". Biochimica et Biophysica Acta (BBA) - General Subjects. 1723 (1–3): 19–32. doi:10.1016/j.bbagen.2005.02.003. PMID   15777735.
  28. 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.
  1. ↑ Beebe JA, Frey PA (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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