Lactase

1. Lactase An Important Biological Enzyme By: Melissa Appio

2. Enzymes An enzyme is a protein that catalyses a chemical reaction. Enzymes are able to speed up the reaction by lowering the activation energy needed for the reaction to occur.

3. Lactose Lactose itself is a disaccharide, a compound sugar made up from two simple sugars… Galactose and Glucose.

4. Lactase as an Enzyme... Lactase, also known as beta-galactosidase, is the enzyme that catalyzes the hydrolysis of lactose.

5. The Role of Lactase in Humans • In the normal human, lactase in a pivotal enzyme that can be found on the brush border of the small intestine. • Lactase is needed to break down lactose into glucose and galactose. Glucose is the usable form of sugar required by the body The galactose is converted into even more glucose to be used by the body. • Lactose is a sugar is found in many food products, especially dairy products such as milk, cheese, and ice cream.

6. Lactose Intolerance • When there is a lack of the enzyme lactase in the small intestine, a condition known as lactose intolerance results. • Without a sufficient amount of lactase, a patient will not be able to break down all of the lactose he or she ingests. As a result, digestive difficulties become evident.

7. Lactose Intolerance Symptoms • Symptoms tend to vary in severity depending on the amount of lactase a person produces as well as their age, ethnicity, and digestive rate, but common symptoms include… • Intestinal Cramping • Gas • Diarrhea • Nausea • Bloating

8. Lactose Intolerance Diagnosis • The Lactose Tolerance Test • After fasting, the patient must drink a liquid that contains lactose. • The patient’s blood sugar levels are then monitored. • The blood sugar levels indicate how well the lactose is broken down by lactase into glucose (measured by the blood sugar) and galactose. • The Hydrogen Breath Test • Normally there is very little hydrogen in a person’s breath • Yet, undigested lactose can be fermented by bacteria to produce hydrogen gas. • The gas is absorbed by the intestines and carried through the bloodstream to the lungs where it will be exhaled.

9. Lactose Intolerance Alternate Diagnosis Methods Used in Young Children • Stool Acidity Test • When a infant is lactase deficient, the undigested lactose becomes fermented by bacteria in the colon, creating lactic acid and other fatty acids that can be measured in the child’s stool. • Also present in the stool sample in these cases is glucose since the absorbed lactose can be present in the stool sample. • The glucose would be present in the stool sample as part of the lactoes if the lactase was not able to break it down in the intestine. • If the lactose was broken down, the glucose would have been used by the body and not excreted as part of the lactose.

10. Lactose Intolerance Causes • Primary lactase deficiency • This deficiency is a result of the humans producing less and less lactase after the age of two years. • There is a genetic component in many of these cases, and a blood test can be used to identify the presence of the problematic gene. • Secondary lactase deficiency • This deficiency is a result of injury to the intestine as occurs in patients with intestinal diseases such as Crohn’s disease and Celiac Disease. • When these diseases are under control, the lactase comes back in normal functioning and digests the lactose.

11. Lactose Intolerance Treatment • When the body fails to produce enough lactase in the intestines to break down the lactose ingested with a person’s meals, the person can avoid unpleasant symptoms by avoiding products containing lactose. • Yet, products containing lactose are also the products high in calcium. Denying one’s body of the calcium they need would be detrimental to the person’s health. • Therefore, artificial forms of lactase can be taken in pill/ capsule form or added directly to the food. These medications contain the lactase enzyme so that lactose can be digested in these patients.

12. Now Focusing on... Lactase & Its Normal Functioning • Lactase is needed in order for many biological processes to occur. • First lactase breaks down the lactose into glucose and galactose. • The glucose is then used for glycolysis. • During glycolysis, glucose is oxidized to two molecules of pyruvic acid, yielding 2 ATP of energy. • Glycolysis occurs in the cytoplasm of cells, not in organelles, and occurs in all kinds of living organisms.

13. Lactase in E. coli • The enzyme lactase is not only found in humans. It is involved in many biological processes in other living things as well. • For example, lactase can be found in Escherichia coli where it is a product of the Z gene of the lac operon of E. coli. • Here lactase hydrolyzes the disaccharide lactose to galactose and glucose in addition to converting the lactose into another disaccharide, allolactose. • Allolactose is the natural inducer for the lac operon.

14. Lactase Homotetramer

15. Reaction Being Studied Now I will break down the steps involved...

16. Lactase in complex with Lactose • The initial binding of a lactose substrate to the lactase enzyme is observed based on the stacking on tryptophan (Trp999). • The stacking of networks of specific interaction made by galactsyl hydroxyls occurs on the Trp 999 residue before catalysis can occur.

17. Once the substrate is aligned in the correct position, then a glutamic acid (Glu 461) can act as an acid and donate a proton to a glycosidic oxygen. • The glycosidic oxyge is involved in the formation of an intermediate with another glutamic acid (Glu 537). • Then a base enters in the reaction to assist in the release of the intermediate. This base is also the Glu 461 which can now act as a base since it has lost protons in the pervious step. • Acting now as a base, Glu 461 accepts from the acceptor molecule, which is an alcohol, R’OH in this case.

18. The formation of the covalent intermediate acts to neutralize the charge on the nucleophile Glu537. • Because of this close proximity of Glu 537 and Glu 461, the neutralization of the Glu537 decreases the pKa of Glu 461. • Glu 461 also interacts with a magnesium ion that it believed to assist in this lowering of pKa. • With this decrease in pKa, the Glu461 is more likely to be deprotonated. • Since it is has lost protons when it was deprotonated, the Glu461 can act as a base in the next step accepting the protons it originally lost protons to replace the ones it lost.

19. Now in the second half of the reaction, there is a galactosyl transfer from Glu 537 to an acceptor molecule. • As a result of this transfer, Glu 537 becomes more negatively charged. • This negative charge formation is facilitated by partial proton donation of a tyrosine (Tyr 503).

20. At this point the lactose hydrolyzes and breaks off the active site revealing glucose and galactose. • In place of the substrate, a water molecule hydrogen bonds to Glu 461 and to the ring of the galactsyl moiety. • Sometimes the glucose molecule does not diffuse away because it interacts with asparagine (Asn 102) and histidine (His 418) since it is covalently bonded deep into the active site. • As a result, the hydroxyl with the longest connection to the pyranose ring, in this cases allolactose, has the best chance of releasing this intermediate that was formed. • In this process of transglycosylation, which is the transfer of a glycosidically bound sugar to another hydroxyl group, allolactose, or galaactsyl 1-6 glucose, is the preferred product.

21. End Products • As a result of the hydrolysis, the enzyme lactase is able to break down its substrate lactose into glucose and galactose. • In addition lactase converted the lactose into another disaccharide, allolactose. • Now, the glucose is available for use by the cell.

22. Works Cited Sites Juers, Douglas H. , Tom D. Heightman, Andrea Vasella, John D. McCarter, Lloyd Mackenzie, Stephen G. Withers, and Brian W. Matthews. “A Structural View of the Action of Escherichia coli (lacZ) –Galactosidase.” Institute of Molecular Biology, Howard Hughes Medical Institute and Department of Physics, University of Oregon, Eugene, Oregon 97403-1229, Laboratorium für Organische Chemie, ETH-Zentrum, Universitätstrasse 16, CH-8092 Zürich, Switzerland, and Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1, 2001. “Lactase.” Science Projects Online, 2001. http://www.science-projects.com/Lactase.htm “Lactase – Beta-galactosidase” China GreatVista Chemicals, 2005. http://www.greatvistachemicals.com/biochemicals/lactase.html “Lactose Intolerance - NIH Publication No. 06–2751 National Institute of Health – National Institute of Diabetes and Digestive and Kidney Diseases, March 2006. http://digestive.niddk.nih.gov/ddiseases/pubs/lactoseintolerance/ McKusick, Victor A. “Lactase – LCT” OMIM - Online Mendelian Inheritance in Man. John Hopkins University, 2006. Montalto, Massimo, Valentina, Curigliano, Luca Santoro, Monica Vastola, Giovanni Cammarota, Raffaele Manna, Antonio Gasbarrini, Giovanni Gasbarrini. “Management and Treatment of Lactose Malabsorption” World Journal of Gastroenterology. Department of Internal Medicine, Catholic University, Rome, Italy, 2005. “A Primer on Photosynthesis and the Functioning of Cells.” Introduction to Global Change. University of Michigan. http://www.globalchange.umich.edu/globalchange1/current/lectures/kling/energyflow/PSN_primer.html

23. Works Cited Images sp.uconn.edu/~terry/images/micro/.finderinfo www.asanono.com/medical/intestines_800.jpg www.astrographics.com/GalleryPrints/Display/GP2144.jpg www.lclark.edu/~bkbaxter/.../apr_2.htm www.co.mohave.az.us/WIC www.moomilk.com/archive/99_images/july99_feat.jpg www.nobodysells4less.com/lactaid-ultra.htm www.shopndeals.com/