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Georgia Lofts

Georgia Lofts


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About Me:Biomedical Science Graduate

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Enzyme experiment part 2

Enzyme experiment part 2

Yeast pyruvate kinase activity can be altered through the use of allosteric regulators


Metabolic pathways are highly controlled with several control points which regulate enzyme activity. Glycolysis and gluconeogenesis have predominantly the same enzymes, one being pyruvate kinase, and they are controlled by the concentrations of substrates and products. Experimental procedure can demonstrate the action of allosteric effectors FBP, ATP and alanine on pyruvate kinase. Using a spectrophotometer, absorbance values can be taken to show the rate of activity, thus demonstrating the effect of regulators. Regulators vary in their significance for enzyme activity, FBP has the most significant impact on pyruvate kinase. FBP increases pyruvate kinase activity whereas ATP and alanine are inhibitors, thus decrease pyruvate kinase activity.


Allosteric regulation is when an enzymes activity is effected by an effector molecule known as an allosteric regulator. The effector binds to a site known as the allosteric site, meaning a part of the enzyme that is not the active site. Allosteric regulators catalytic activity is controlled by the noncovalent binding of specific effector molecules. Rapid changes in enzyme activity occur when allosteric regulators are present, especially through changes in concentration of effectors. The flow of metabolites in pathways are also controlled by allosteric regulators. Allosteric enzymes play a key role in ensuring that glycolysis and gluconeogenesis do not occur at the same time. They also prevent an excessive build-up of certain molecules. Pyruvate kinase is an enzyme which functions in regulating and coordinating glycolysis and gluconeogenesis. The enzyme activity leads to the yielding of one molecule of pyruvate and one molecule of ATP. Positive allosteric activators increase enzyme activity whilst negative allosteric activators decrease activity.


Allosteric regulation of pyruvate kinase is important to meet two major cellular needs; the breakdown of glucose to generate ATP and the provision of building blocks for biosynthesis. Pyruvate kinase activity is essentially irreversible; thus, it becomes more or less active in response to allosteric binding.


Fructose 1,6-bisphosphate (FBP) is an intermediate of glycolysis that regulates pyruvate kinase activity allosterically. Pyruvate kinase is activated by FBP, then FBP is converted into the substrate PEP. This is known as feed-forward activation, the term is used to describe FBP’s effect on pyruvate kinase and acts to start the pyruvate kinase reaction. Adenosine triphosphate (ATP) is a nucleotide that is composed of the nitrogenous base adenine, the sugar ribose and three phosphate groups. It is an essential energy carrier in all living organisms. ATP not only functions in providing energy for cells, but also is an allosteric regulator of pyruvate kinase. Alanine is an amino acid which is used in the biosynthesis of proteins. It is also an allosteric regulator of pyruvate kinase. The aim of this experiment is to explore the action of allosteric regulators FBP, ATP and alanine through determining the protein concentration of the extract. Using the molar extinction coefficient for NADH, pyruvate kinase activity can be expressed in absolute units.


The experimental procedure consists of using allosteric regulators FBP, ATP and alanine to measure the effects of their presence on the activity of yeast pyruvate kinase. Absorbance values will be measured using a spectrophotometer, at 340nm in wavelength, to show the rate of activity. Using the molar extinction coefficient for NADH, the reaction rate can be expressed in absolute units.



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