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Glycosidase Problem Space

Plants with high starch content such as corn, potatoes, rice, sorghum, wheat, and cassava have played significant roles in human development. From hamanch (manioc beer) to high fructose corn syrup, both low tech and high tech approaches to the use of starch prevail.

Starch molecules are glucose polymers linked together by the alpha-1,4 and alpha-1,6 glycosidic bonds. A family of enzymes known as the glycosidases aid in the breakdown of starch and other glucose polymers such as celluslose and glycogen to smaller sugars, and finally, to glucose.

All of these enzymes hydrolyze glycosidic bonds, but some are also multifunctional. Sequence data for many of the glycosidases are well described in terms of their functional roles (active sites and protein folding) and a great deal of research can be found on evolutionary relationships between these enzymes in different taxa.

Not only alpha-amylases, but also beta-amylases and starch debranching enzymes such as the pullulanases and glucosidases also contain the beta/alpha barrel domain including catalytic amino acids. The mechanisms differ, but the relatedness of these enzymes is clear. Amino acid sequences of the beta-strands are well conserved within this family of enzymes. This provides a rationale for using the glycosidases to investigate the evolutionary relationships between organisms.

Glycosidases and the modification of corn starch

In the commercial production of maltodextrins and corn syrups, starch is hydrolyzed using an alpha-amylase either alone or combined with other enzymes.

  • Maltodextrins are partially hydrolyzed starches used in foods to modify physical properties that contribute little or no sweetness or flavor. Alpha-amylase is used to make this product.
  • Corn syrups are used primarily to add sweetness or enhance flavors in food products. High dextrose syrup is made by hydrolyzing starch first with alpha-amylase , then with glucoamylase (amyloglucosidase ) which cleaves both alpha-1,4 bonds and alpha-1,6 bonds. To increase the rate of alpha-1,6 bond cleavage, a debranching enzyme such as pullulanase may also be added.

    High fructose corn syrup is made by converting dextrose to fructose using glucose isomerase (not a glycosidase) to create an equilibrium mixture of dextrose and fructose (42%fructose). Higher fructose concentrations can be prepared by separating fructose from dextrose using chromatographic methods and large-scale ion exchange columns. Pure crystalline fructose is made this way.

What does a glycosidase look like?

The alpha-amylases contain eight alpha-helices and eight beta-strands in beta alpha/beta alpha order. The alpha-helices provide rigidity to the catalytic sites and substrate binding sites which are contained within the beta-strands.

 

See also: Alpha/Beta Topologies http://www.cryst.bbk.ac.uk/PPS95/course/8_folds/alph_bet_wnd.html#barrels

How does a glycosidase break down starch?

If we look at the alpha-amylase enzyme, we can find both the catalytic sites and the substrate binding site. The amino acid sequence of alpha-amylases may vary, but there are specific aspartic acid and glutamic acid units found in the beta-strand region that are responsible for the catalysis of glycosidic bond cleavage. Other amino acid units such as histidine are necessary for enzyme activity involved in establishing conformation and binding of the substrate.

Steps in enzymatic hydrolysis of starch

  1. Acid catalyzed nucleophilic displacement. One aspartic acid acts as the nucleophile, while the other aspartic acid is the acid catalyst. Two histidine amino acids form hydrogen bonds to the substrate to hold it in place.
  2. Acid catalyzed hydrolysis of the link between the substrate polysaccharide and the enzyme (a carbohydrate protein ester link).
  3. The end products are the two fragments of the substrate polysaccharide and the freed enzyme.

Conservation of active sites and catalytic sites

Not only alpha-amylases, but also beta-amylases and starch debranching enzymes such as the pullulanases and isoamylases also contain the beta/alpha barrel domain including the same catalytic amino acids. The mechanisms differ, but the relatedness of these enzymes is clear. Amino acid sequences of the beta-strands are well conserved within this family of enzymes. This provides a rationale for using the glycosidases to investigate the evolutionary relationships between organisms.

Solving the problem of synonyms

Besides providing essential information on enzyme classification, E.C. numbers are very useful for doing searches when variations of enzyme names are encountered.

Enzyme functions are classified by E.C. numbers:

  • E.C.1. Oxidoreductases.
  • E.C.2. Transferases.
  • E.C.3. Hydrolases.
  • E.C.4. Lyases.
  • E.C.5. Isomerases.
  • E.C.6. Ligases.

    (Enzyme Data Bank)

Listed below are the E.C. numbers of several starch hydrolyzing enzymes further characterized by their mode of action:

  • Alpha-amylases (EC number 3.2.1.1) hydrolyze starch by cleaving alpha 1,4 linkages randomly within the chain (endo mechanism)
  • Beta-amylases (EC number 3.2.1.2) hydrolyze starch by cleaving alpha 1,4 linkages producing maltose units from the non-reducing end ( exo mechanism)
  • Amyloglucosidase s (EC number 3.2.1.33) hydrolyze starch by cleaving glucose units from the non-reducing end (exo)
  • Pullulanases (EC number 3.2.1.41) and isoamylases (EC number 3.2.1.68) are debranching enzymes that hydrolyze starch by cleaving alpha 1,6 linkages (specific so not referred to as either endo or exo)

 

Visualization

Structural data files for many of the glycosidases are readily available from the Protein Data Bank. All pdb files have unique 4 character names that include numbers and letters. The Protein Data Bank Education page provides a good introduction to the international repository for 3-D molecular structure data.

See: http://www.rcsb.org/pdb/education.html

“Our vision is for the PDB to enable scientists worldwide to gain a greater understanding of structure-function relationships in biological systems," Helen Berman, Rutgers , is principal investigator for the PDB project.

Data

Amylase FASTA Sequences

Tools

PDB Viewers, RasMol program and VRML browser
http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/

Biology Workbench
http://workbench.sdsc.edu

ConSurf Server
http://consurf.tau.ac.il/

PyMOL
http://pymol.sourceforge.net/

RSCB Protein Data Bank
http://www.rcsb.org/pdb/

Summaries and structural analyses of PDB data files
http://www.biochem.ucl.ac.uk/bsm/pdbsum/index.html

Protein Explorer
http://www.umass.edu/microbio/chime/explorer/

“Protein Explorer can make visual exploration of protein structure much more accessible to novices, occasional users, or nonspecialists, as well as making it much more convenient than RasMol, even for experts.”

You can use this viewer by directly entering the pdb file name or by setting up a web page of pdb file name links that you are interested in. This viewer can also be used off line with downloaded pdb files.

Potential Investigations

In addition to introductory Microbes Count! Activities, the following scenarios are presented:

Scenario 1. Glycosidases and the modification of corn starch …

In the commercial production of maltodextrins and corn syrups, starch is hydrolyzed using an alpha-amylase either alone or combined with other enzymes.

Maltodextrins are partially hydrolyzed starches used in foods to modify physical properties that contribute little or no sweetness or flavor. Alpha-amylase is used to make this product.

Corn syrups are used primarily to add sweetness or enhance flavors in food products. High dextrose syrup is made by hydrolyzing starch first with alpha-amylase, then with glucoamylase (amyloglucosidase) which cleaves both alpha-1,4 bonds and alpha-1,6 bonds. To increase the rate of alpha-1,6 bond cleavage, a debranching enzyme such as pullulanase may also be added.

High fructose corn syrup is made by converting dextrose to fructose using glucose isomerase (not a glycosidase) to create an equilibrium mixture of dextrose and fructose (42%fructose). Higher fructose concentrations can be prepared by separating fructose from dextrose using chromatographic methods and large-scale ion exchange columns. Pure crystalline fructose is made this way.

  • List an organism that is the source of one of these industrial glycosidase enzymes above. Does it produce starch?
  • Industrial corn starch processing occurs in water heated to 90 degrees C. The use of thermostable enzymes is required. Where would you expect to find an amylase that could be used?
  • Choose a food product containing high-fructose corn syrup other than a can of pop. Create a poster showing the role of glycosidases in production of the product.

Resource: http://home3.inet.tele.dk/starch/ industrial starch processing

Scenario 2. Enzyme replacement therapy: Should you try increasing your own levels of alpha-amylase?

There are a number of over-the-counter products that contain enzymes that aid in the digestion of proteins, starches, fats and dairy foods. For example, Lactaid® contains the enzyme lactase for helping the digestion of dairy products.

 
  • Do you have any concerns about enzyme replacement therapy? Explain.
  •  

Another commercial product, Digestol®, is advertised as an all-purpose digestive aid. http://www.kramerlabs.com/index.asp?template=digestol

The product contains the following enzymes:

  • Support or reject claims made by the manufacturer on the efficacy of this diet aid. Provide evidence and identify your sources.
  • How might you use bioinformatics to convince others that all amylases are not the same? Choose three and present your case.  

Scenario 3. Alpha-amylase inhibitors, weight loss, and beans

When trying to lose weight, dieters limit the amount of starch in their diet. The usual amounts of starchy foods, such as potatoes, bread, beans, corn and pasta, are reduced. Starch provides from 500 to 700 calories per day in the average American adult diet. A gram of starch, when digested and absorbed, provides 4 calories. Individuals may consume as much as 1,500 or more calories per day from starch contained in their foods. However, starch is a large molecule that cannot be absorbed if it is not first broken down. Undigested starch will pass on through the digestive tract. Plant extracts containing AIU's (alpha-amylase inhibiting units) such as phaseotein from legumes are said to inhibit the absorption of starch by blocking the enzyme alpha-amylase.

  • Find an over-the-counter product used to block starch digestion. Describe the product.
  • Would you take alpha-amylase inhibitor (AIU) tablets in order to lose weight? Explain.
  • How do AIU's work?
  • Do bean plants make the alpha-amylase inhibitor (AIU) phaseotein in order to lose weight? Provide an alternative explanation.  

Scenario 4.   Allergic to your breakfast cereal? Is the alpha-amylase inhibitor in wheat the culprit?

Using serum samples collected from children with a known wheat allergy and one adult with baker's asthma, a wheat protein was identified which bound IgE. Control serum samples were collected from wheat-tolerant patients. No IgE binding to this wheat protein was demonstrated in any of the control subjects.

Samples representing the 15 kd wheat protein (isoelective point 5.85) were selected and the N-terminal peptide sequence of this protein (residues 1 to 20) matched to a wheat alpha-amylase inhibitor.

  • Could you use this sequence to search for similar alpha-amylase inhibitors in other grains? Explain.
  • Develop a poster for this research that would be appropriate for public education about wheat allergy at a children's health center.  

Resource: James JM, Sixbey JP, Helm RM, Bannon GA, Burks AW. 1997. Wheat alpha-amylase inhibitor: a second route of allergic sensitization. Journal of Allergy Clinical Immunology. 99(2): 239-44

Scenario 5. Investigating the human alpha-glucosidase gene

Glucose is a major source of energy for the body. It is stored in the form of glycogen in both the liver and muscles and later released with the help of enzymes. Persons affected by glycogen storage disease (GSD) have an inherited defect in one of the enzymes responsible for forming or releasing glycogen as it is needed by the body during exercise and/or between meals. There are eleven types of GSD known at this time.

Read the following brochure written by a mother whose son inherited an infantile form of Pompe's Disease which reduces glycogen storage function to less than 2% of normal. This is an autosomal recessive disorder that is always fatal.

See: http://www.pompe.org.uk/agsdpom.html   Pompe's Disease: A Guide for Families

  • Construct a family pedigree to use to explain the genetic basis of this disorder.
  • Choose two known alpha-glucosidase mutations and explain why the enzyme doesn't function normally.

Resources

Clinical Genetics Site
http://www.eur.nl/FGG/CH1/pompe/

NiceZyme View of ENZYME: EC 3.2.1.3
http://www.expasy.ch/cgi-bin/nicezyme.pl?3.2.1.3

GSD II Database: A register of mutations in Human acid alpha-glucosidase
http://www.eur.nl/FGG/CH1/pompe/mutation.htm

Looking into Glycosidases: A Bioinformatics Resource for Biology Students
Editable Word Doc (2.3 MB)      PDF Version (1 MB)

Starch: Chemistry and Technology. Whistler, BeMiller & Paschall, Eds. 1984. San Diego : Academic Press.

Starch: Properties and Potential, Galliard, Ed. 1987. New York : John Wiley & Sons.

Note: Names used for this disease:

Glycogen Storage Disease Type II (GSD II)
Acid Maltase Deficiency
Pompe Disease
Lysosomal alpha-glucosidase Deficiency

Author: Ethel Stanley 2005