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Amanda Hensley's Protein

Science Building
CPO 2191
859-985-3318

Office Hours:
M–F, 8:00 a.m.–5:00 p.m.


Advanced Biochemistry CHM486A
Science Building Room 306
MWF, 10:00a.m.–11:00a.m.
Instructor: Dr Matthew Saderholm

Contact:
Anthony Reynolds_at_berea.edu

The Enzyme Beta-Galactosidase: Structure and Function
by Amanda L. Hensley


What is an Enzyme?
          An enzyme is a complex that helps to speed reactions. In the case of galactosidase, the enzyme is a protein, made up of amino acids. These are reactions that, given enough time, would occur naturally. However, the amount of time is often years. Many biological reactions, like blood clotting or an organism obtaining nutrition from its food would be practically impossible without the help of enzymes.

Lactose, the substrate of beta-galactosidase
(Bungay, 1997)
        


What is a Substrate?
          A substrate is the molecular compound for which an enzyme catalyzes, or speeds up, a chemical reaction. Lactose is a sugar that exists mostly in dairy products. It is a disaccharide, meaning that it is made up of two sugars, made up of one unit of galactose and one unit of glucose in a one to four linkage. In order for organisms to use the sugar, taking advantage of glycolysis, the Krebs cycle, or fermentation, the two units must be hydrolyzed, using water to break them apart. To rend the simple sugars organisms utilize a protein enzyme; in animal systems this enzyme is called lactase, and in bacterial systems it is galactosidase. Although this enzyme also utilizes other substrates, such as allolactose, the focus will be on its catalytic affect on lactose.

What is Autology?
          When two proteins do not arise from the same lineages but have the same function they are said to be autologous. The lactose in humans and the galactosidase in bacteria are autologous (Fabre et al, 2008). This can be seen by comparing the functions and the substrates of the two enzymes. This can be seen by comparing the functions of the two enzymes. In this case, both enzymes cleave lactose through hydrolysis, using water to break the bonds.

What is Sequence Conservation?
          Every time DNA is replicated, mistakes are made and an individual base, such as adenine, may be replaced with another, such as guanine. Over time mistakes in the genetic sequence that codes for a certain protein add up, giving proteins in slightly different amino acid sequences. If you check the Spacefill and then Consurf A boxes on the three-dimensional protein visualization, it will show you how much of this enzyme is conserved across known forms of this lactase. It will only show one of the four identical subunits, but they are all equally conserved, and in the same regions. The red amino acids are rarely conserved, and the for the rest of the amino acids, the darker the blue, the more they are conserved. If you also check the Slab button, you can click on the protein and pull it around, viewing all of the interior amino acids. You will notice that the inside of the active site, where the reactions take place, is much more conserved (more and darker blue) because these amino acids are what make the enzyme functional and must be conserved for the enzyme to hydrolyze properly.

Galactosidase structure
          The galactosidase present specifically in Escherichia coli is a tetrameric enzyme that contains four identical sidechains of 1021 amino acids each (Zabin, 1982). The number of amino acids varies slightly. The four subunits are identical and the overal enzyme is rectangular, with each subunit having an active site. By clicking the 2nd Structure/ Color by amino acid box, the structure of the protein can be seen in terms of the constituent secondary structures. The yellow ribbons are beta sheets, and the pink curls are the alpha-helices. In biological systems, these proteins are always in complex with water. To see where it would orient, check the Show Water box.

Mechanism
The mechanism for how the enzyme actually catalyzes this reaction includes non-covalent interactions, general acid/base catalysis, and electrostatic/covalent stabilization (Juers, et al., 1994). The enzyme has developed to prefer speed over binding the substrate tightly. The acid/base component, using the pH, work to stabilize the intermediate forms of the substrate, and seems to rely heavily on the Tyr-503 (the Tyrosine, an amino acid, that is the 503rd in the chain of the protein). Magnesium may be acting as a eletrophilic catalyst (Selwood and Sinnott, 1990). The electrostatic/covalent catalysis suggests that there is at least a galactosyl cation transition state that is stabilized by Glu-461 (Glutamic acid that is 461st in the chain). This residue also seems to induce the formation of a cation by electrostatic stabilization (Huber et al, 1993). Skipping the complex intermediates, the following picture shows how water is used, illustrating the initial and final products.

Schematic of the addition of water to reduce lactose to
glucose and galactose (Logbuch, 2004)
        


Lactose Intolerance
          In humans, the absence of the lactase enzyme can cause problems digesting dairy. In such individuals, the symptoms can include abdominal cramps, diarrhea, and excessive gas after consuming dairy products. Also, the expression of lactose enzymes can also be lost in individuals who do not consume dairy for long periods of time. The problems that are encountered with lactose can be prevented by either avoiding dairy foods or taking an enzyme supplement with meals rich in dairy products.

Practical Uses of Galactosidase
          Galactosidase has several practical uses. The dairy industry would not be at all what it is without the ability to ferment the sugars from lactose. The bacteria that culture yogurts, cheeses, sour cream, and many other products are only viable because of this enzyme. Recently, the enzyme has been isolated from bacterial systems to be added into finished dairy products to break down the remaining lactose so that they are lactose-free and do not cause digestive disruption to those suffering from lactose intolerance. The presence or absence of this enzyme is also a crucial diagnostic test in determining whether bacteria present in the water supply are coliform bacteria, indicating the presence of feces and inadequate sanitation. Galactosidase is present in Escherichia coli, a common fecal bacterium.
          Because of the autology of beta-galactosidase and human intestinal lactase, studies suggest that it could also be a promising candidate for use in gene replacement therapy, inserting the bacterial DNA into a human genome so that individuals suffering from lactose intolerance would produce their own lactase (Fabre, et al, 2008).

Conclusions
          Galactosidase is a necessary enzyme to biological systems as a means of utilizing sugars for energy. Through understanding its form and function, additional uses of this enzyme become clear through research, including medical and biochemical uses.







References
1. Bungay, H.R. (1997) Higher sugars and polymers. rpi.edu (obtained digitally)

2. Fabre, J., Salehi, S., Eckley, L.,Sawyer, G., Zhang, X., Dong, X., Frud, J.N. (2008) Intestinal lactase as an           autologous B-galactosidase reporter gene for in vivo gene expression studies. Human Gene Therapy.           (submitted Aug 22, 2008)

3. Zabin, I. (1982) B-galactosidase a-complementation- A model of protein-protein interaction. Molecular and           Cellular Biochemistry, 49 (2), 87-96. (ordered from interlibrary loan)

4. Juers, D.H., Heightman, T.D., Vasella, A., McCarter, J.D., Mackenzie, L., Withers, S.G., Matthews, B.W. (2001) A           structural view of the action of Escherichia coli lacZ B-galactosidase. Biochemistry, 40 (49) 14781-14794.           (obtained from Scopus)

5. Selwood, T., and Sinnott, M.L. (1990) The solvent-isotope-effect study of proton transfer during catalysis by           Escherichia coli (lacZ) B-galactosiase. Biochem. J. 268, 317-323.

6. Huber, R.E., Wallenfels, K., Kurz, G. (1975) The action of B galactosidase (Escherichia coli) on allolactose.           Canadian Journal of Biochemistry, 53 (9), 1035-1038. (obtained through interlibrary loan)

7. Logbuch, L. (2004) Laktose. German wikipedia project. Obtained from           http://commons.wikimedia.org/wiki/Image:Lactose_color.png