Enzymes Specificity

Enzymes Specificity
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Purpose: This experiment seeks to check the ability of lactase to specifically interact and bind to lactose as opposed to maltose.
Background
The experiment seeks to test enzyme specificity for the lactase enzyme on two separate substrates of maltose and lactose. In definition, an enzyme is a chemical catalyst that helps in speeding up a chemical reaction by reducing the required activation energy to facilitate the reaction ADDIN CSL_CITATION { “citationItems” : [ { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1002/9780470015902.a0000716.pub2”, “ISBN” : “9780470015902”, “ISSN” : “9780470015902”, “abstract” : “Speciufb01city is a hallmark of enzyme catalysis; it is inseparable from catalytic eufb03ciency, the other hallmark of enzyme reactions. Speciufb01city arises from the three- dimensional structure of the enzyme active site; this site is complementary to the transition state of the reaction. The substrate ufb01ts snugly within the enzyme active site, optimally aligned to react with the catalytic residues. In addition, the conformation of the substrate is constrained, which will control the course of the reaction.”, “author” : [ { “dropping-particle” : “”, “family” : “Hedstrom”, “given” : “Lizbeth”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } ], “container-title” : “Encyclopedia of Life Sciences”, “id” : “ITEM-1”, “issued” : { “date-parts” : [ [ “2010” ] ] }, “title” : “Enzyme Specificity and Selectivity”, “type” : “chapter” }, “uris” : [ “http://www.

mendeley.com/documents/?uuid=b611d963-1182-4432-9657-db5323bc00ec” ] } ], “mendeley” : { “formattedCitation” : “(Hedstrom, 2010)”, “plainTextFormattedCitation” : “(Hedstrom, 2010)”, “previouslyFormattedCitation” : “(Hedstrom, 2010)” }, “properties” : { }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }(Hedstrom, 2010). In order for the reaction to take place or be sped up, the substance on which the enzyme acts on, also referred to as the substrate, has to bind in the active site(s) of the enzyme ADDIN CSL_CITATION { “citationItems” : [ { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1002/9780470015902.a0000716.pub2”, “ISBN” : “9780470015902”, “ISSN” : “9780470015902”, “abstract” : “Speciufb01city is a hallmark of enzyme catalysis; it is inseparable from catalytic eufb03ciency, the other hallmark of enzyme reactions. Speciufb01city arises from the three- dimensional structure of the enzyme active site; this site is complementary to the transition state of the reaction. The substrate ufb01ts snugly within the enzyme active site, optimally aligned to react with the catalytic residues. In addition, the conformation of the substrate is constrained, which will control the course of the reaction.”, “author” : [ { “dropping-particle” : “”, “family” : “Hedstrom”, “given” : “Lizbeth”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } ], “container-title” : “Encyclopedia of Life Sciences”, “id” : “ITEM-1”, “issued” : { “date-parts” : [ [ “2010” ] ] }, “title” : “Enzyme Specificity and Selectivity”, “type” : “chapter” }, “uris” : [ “http://www.mendeley.com/documents/?uuid=b611d963-1182-4432-9657-db5323bc00ec” ] } ], “mendeley” : { “formattedCitation” : “(Hedstrom, 2010)”, “plainTextFormattedCitation” : “(Hedstrom, 2010)”, “previouslyFormattedCitation” : “(Hedstrom, 2010)” }, “properties” : { }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }(Hedstrom, 2010). This substrate should have a specific structure that would help it fit into the enzyme’s active site to facilitate the reaction catalysis. In other words, a specific substrate can only bind to a particular enzyme. For instance, enzyme lactase is only specific to the lactose substrate. Lactose is one of the disaccharides, and it is found in dairy milk products, and it is made up of two monosaccharides namely; glucose and galactose. Another substrate known as maltose is also made up of two glucose monosaccharides. Maltose and lactose are relatively similar in structure. When enzyme lactase is added the two substrates to check the specificity that the substrate requires to bind to the substrate despite their relatively similar structures. Therefore, the glucose amount was measured, to ascertain the substrate that was specific to enzyme lactase. Glucose was measured for both reactions to check whether the effect of lactase in catalyzing the hydrolysis reaction. The one that was found to have more glucose was the one that was most specific to the lactase enzyme.
Hypothesis: Knowing that lactase is specific to lactose when it is added to maltose and lactose, enzyme lactose will bind to the lactose substrate and hasten its metabolism than it will on the maltose substrate, hence, more glucose will be produced from the lactose substrate.
Methods and Methods-Specificity Experiment
Label lactose and maltose microfuge tubes L and M respectively.
With the aid of a sterilized plastic pipette, put the substrate (milk) to the 0.5 mark on the centrifuge tube L. The substrate in this tube has to be 500 µL.
With the help of a clean pipette, put maltose solution into the M tube up to the 0.5 mark. The volume of the maltose has to be 500 µL.
Then use the clean pipette to put lactase solution to both microfuge tubes, up to the 1.0 mark. Both tubes have to contain 1.0 mL of the mixture composed of 500 µL maltose or milk and 500 µL of the enzyme lactase.
Incubate the tubes for 10 minutes in 40º C water bath.
After the 10 minutes are over, put a glucose strip in both tubes. After about a second, remove the strip and put it on the bench for half a minute. After that, compare the color of the strips to the provided chart. This chart will help show the glucose content in mg/dL.
Results
Substrate Lactose Maltose
1 250 0
2 250 0
3 500 0
4 2000 100
5 500 0
Discussion
In the above experiment, it is clear that the lactase enzyme managed to break down lactose into glucose and galactose. The breakdown of lactose is shown by the addition of the lactase enzyme to the milk solution. After adding the enzyme, glucose was found in virtually all the used centrifuge tubes (250, 250, 500, 2000, 500 mg/dL). The presence of glucose was attested by the change of coloration in the used strips. The specificity of the lactase enzyme was demonstrated by its reaction to lactose in particular. When added to the maltose solution, the enzyme did not catalyze the reaction to break down it into two glucose molecules as exhibited by the zero readings. Errors were seen in solution four, which might be attributed to inaccurate measurements.
References
ADDIN Mendeley Bibliography CSL_BIBLIOGRAPHY Hedstrom, L. (2010). Enzyme Specificity and Selectivity. In Encyclopedia of Life Sciences. https://doi.org/10.1002/9780470015902.a0000716.pub2