Essential idea: Enzymes control the metabolism of the cell. Above is just a small part of the IUBMB-Sigma-Nicholson Metabolic Pathways Chart aims to show all the metabolic pathways found in eukaryote cells. The chart in it's entirety shows how complex the chemicals reactions needed to support life in a single cell unit. Every arrow on the chart shows an enzyme controlling the conversion of compounds. Edited by Emrich/Black

Understandings, Applications and Skills Statement Guidance 2.5.U1 Enzymes have an active site to which specific substrates bind. 2.5.U2 Enzyme catalysis involves molecular motion and the collision of substrates with the active site. 2.5.U3 Temperature, pH and substrate concentration affect the rate of activity of enzymes. Students should be able to sketch graphs to show the expected effects of temperature, pH and substrate concentration on the activity of enzymes. They should be able to explain the patterns or trends apparent in these graphs. 2.5.U4 Enzymes can be denatured. 2.5.U5 Immobilized enzymes are widely used in industry. 2.5.A1 Methods of production of lactose-free milk and its advantages. Lactase can be immobilized in alginate beads and experiments can then be carried out in which the lactose in milk is hydrolysed. 2.5.S1 Design of experiments to test the effect of temperature, pH and substrate concentration on the activity of enzymes. 2.5.S2 Experimental investigation of a factor affecting enzyme activity. (Practical 3)

Understandings, Applications and Skills Statement Guidance 8.1.U1 Metabolic pathways consist of chains and cycles of enzyme-catalysed reactions. 8.1.U2 Enzymes lower the activation energy of the chemical reactions that they catalyse. 8.1.U3 Enzyme inhibitors can be competitive or non-competitive. Enzyme inhibition should be studied using one specific example for competitive and non-competitive inhibition. 8.1.U4 Metabolic pathways can be controlled by end-product inhibition. 8.1.A1 End-product inhibition of the pathway that converts threonine to isoleucine. 8.1.A2 Use of databases to identify potential new anti-malarial drugs. 8.1.S1 Calculating and plotting rates of reaction from raw experimental results. 8.1.S2 Distinguishing different types of inhibition from graphs at specified substrate concentration.

It is one of the four macromolecules. It is assembled by a ribosome. What is an enzyme? It is one of the four macromolecules. It is assembled by a ribosome. It is composed of amino acids Enzyme: A protein that acts as a biological catalyst lowering the activation energy.

Lowering activation energy barrier Reactants (a) Without enzyme Products Enzyme (b) With enzyme

Lowering Activation Energy Due to the binding the bonds in the substrate molecule are stressed/become less stable

Enzyme Structure http://academic.pgcc.edu/~kroberts/Lecture/Chapter%205/enzymes.html

Enzyme – Substrate Specificity http://droualb.faculty.mjc.edu/Course%20Materials/Physiology%20101/Chapter%20Notes/Fall%202011/chapter_3%20Fall%202011.htm

Lock and Key Model http://www.talkorigins.org/faqs/information/spetner.html

Induced Fit Model http://commons.wikimedia.org/wiki/File:InducedFit_Enzyme_Catalyst.png

Virtual Experience Using the link on Haiku “Interactive Enzyme Virtual Lab” explore how substrates and enzymes come into contact The effect of substrate, temperature, and pH on enzyme action For now disregard anything on inhibitors

Enzymes and temperature

Enyzmes and pH

Enzymes and substrate concentration

Denaturing a protein Enzymes are proteins and denaturation is a key to how enzyme activity is affected by temperature and pH Heat can cause denaturation: vibrations within the molecule breaks intermolecular bonds or interactions. Extremes of pH can cause denaturation: charges on R groups are changed, breaking ionic bonds within the protein or causing new ionic bonds to form. http://upload.wikimedia.org/wikipedia/commons/2/22/Fried_egg%2C_sunny_side_up_%28black_background%29.PNG

For enzymes a change in structure means a change in the active site For enzymes a change in structure means a change in the active site. If the active site changes shape the substrate is no longer able to bind to it. Enzyme before denaturation substrate can bind to the active site Enzyme after denaturation substrate can no longer bind to the active site http://www.biotopics.co.uk/other/enzyme.html

Your aim is to be able not just to recreate the graphs, but to annotate and explain their shape in terms of what is happening at a molecular level.

Common uses of enzymes in industry include: Detergents contain proteases and lipases to help breakdown protein and fat stains Enzymes are widely used in the food industry, e.g. fruit juice, pectin to increase the juice yield from fruit Fructose is used as a sweetener, it is converted from glucose by isomerase Rennin is used to help in cheese production Enzymes are used to breakdown the starch in grains into biofuels that can be combusted Paper production uses enzymes to helping in the pulping of wood In the textiles industry enzymes help in the processing of fibres, e.g. polishing cloth to make it appear more shiny In the brewing industry enzymes help a number of processes including the clarification of the beer In Medicine & Biotechnology enzymes are widely used in everything from diagnostic tests tests to contact lens cleaners to cutting DNA in genetic engineering. http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/Articleimage/2013/CS/c3cs35506c/c3cs35506c-f1.gif

Immobilized Enzymes Enzymes are attached to a material (like glass or gel beads) so that their movement is restricted. Money saving benefits: Enzymes can be used many times since they are easy to separate from the reaction mixture. Easy separation of the enzyme from the product(s). The reaction can be stopped at the proper time. Increased protection of the enzyme against factors that may denature it.

Lactose Intolerance

Production of Lactose-free milk Lactase obtained from commonly from yeast or bacteria. Lactase is bound to the surface of alginate beads Milk is passed (repeatedly) over the beads The lactose is broken down into glucose and galactose The immobilized enzyme remains to be used again and does not affect the quality of the lactose free milk Other uses of lactose free milk: Increases the sweetness of milk Reducing the crystallisation of ice-creams Shortening the production time for yogurts or cheese Other uses of lactose free milk: Increases the sweetness of milk (glucose and galactose are sweeter in flavour) Reducing the crystallisation of ice-creams (glucose and galactose are more soluble than lactose) Shortening the production time for yogurts or cheese (bacteria ferment glucose and galactose more readily than lactose)

Let’s compare some milk from Korea!

TREAD _____ _____ _____ _____ _____ BLINK Metabolic pathways Challenge: by changing just one letter at a time, get from ‘TREAD’ to ‘BLINK’. All intermediates must be real English words. TREAD _____ _____ _____ 8.1.U1 Metabolic pathways consist of chains and cycles of enzyme-catalysed reactions. _____ _____ BLINK

TREAD BREAD BREED BLEED BLEND BLIND BLINK Metabolic pathways Metabolic pathways (biochemical pathways): cycles or chains of enzyme catalysed reactions. TREAD Initial substrate BREAD BREED BLEED intermediates 8.1.U1 Metabolic pathways consist of chains and cycles of enzyme-catalysed reactions. Metabolism: the sum total of all chemical reactions that occur within an organism. Metabolic Pathways: The chemical change from one molecule to another often does not happen not in one large jump, but in a sequence of small steps. The small steps together form what is called a metabolic pathway. BLEND BLIND BLINK end product http://highered.mheducation.com/sites/dl/free/0072437316/120070/bio09.swf

Glycolysis, a part of respiration, is an example of a metabolic chain. Metabolic Pathways Glycolysis, a part of respiration, is an example of a metabolic chain. How many enzymes are involved in this process? biochemmolbio.blogspot.com

Enzyme Inhibition 8.1.U3 Enzyme inhibitors can be competitive or non-competitive. http://www.northland.cc.mn.us/biology/biology1111/animations/enzyme.swf

Inhibitors Inhibitor Substrate Active site Enzyme (b) Enzyme inhibition by a substrate imposter (competitive inhibition) Substrate Active site Enzyme (a) Normal enzyme action (c) Enzyme inhibition by a molecule that causes the active site to change shape (non-competitive inhibition)

Virtual Experience 2.0 Revisit the “Interactive Enzyme Virtual Lab” on Haiku Explore the impact of inhibitors

8.1.U3 Enzyme inhibitors can be competitive or non-competitive.

8.1.U3 Enzyme inhibitors can be competitive or non-competitive.

Example of Non-competitive Inhibition 8.1.U3 Enzyme inhibitors can be competitive or non-competitive.

Features of competitive inhibitors Distinguishing different types of inhibition from graphs at specified substrate concentration. Features of competitive inhibitors When the concentration of substrate begins to exceed the amount of inhibitor, the maximum rate of the uninhibited enzyme can be achieved. However, it takes a much higher concentration of substrate to achieve this maximum rate. Rate of reaction is reduced 8.1.S2 Distinguishing different types of inhibition from graphs at specified substrate concentration. https://wikispaces.psu.edu/download/attachments/46924781/image-6.jpg

Features of non-competitive inhibitors Distinguishing different types of inhibition from graphs at specified substrate concentration. Features of non-competitive inhibitors It takes approximately the same concentration of enzyme to reach the maximum rate, but the maximum rate is lower than the uninhibited enzyme. Rate of reaction is reduced 8.1.S2 Distinguishing different types of inhibition from graphs at specified substrate concentration. The binding of the non-competitive inhibitor prevents some of the enzymes from being able to react regardless of substrate concentration. Those enzymes that do not bind inhibitors follow the same pattern as the normal enzyme. https://wikispaces.psu.edu/download/attachments/46924781/image-6.jpg

End-product inhibition Why might end-product inhibition be useful? What type of inhibition is this?

8.1.U3 Enzyme inhibitors can be competitive or non-competitive. http://highered.mheducation.com/sites/dl/free/0072437316/120070/bio10.swf

Isoleucine is an essential amino acid* End-product inhibition of the pathway that converts threonine to isoleucine. Isoleucine is an essential amino acid* Bacteria synthesize isoleucine from threonine in a series of five enzyme-catalysed steps. As the concentration of isoleucine increases, some of it binds to the allosteric site of threonine deaminase. The pathway is then turned off, regulating isoleucine production. If more isoleucine is used, so its concentration falls, then the allosteric sites of threonine deaminase are emptied and the enzymes restarts the conversion of threonine to isoleucine. 8.1.A1 End-product inhibition of the pathway that converts threonine to isoleucine. *Essential amino acids cannot be made by the body, therefore they must come from food. http://www.uic.edu/classes/bios/bios100/lecturesf04am/feedback-inh.gif

Use of databases to identify potential new anti-malarial drugs. Bioinformatics is an approach whereby multiple research groups can add information to a database enabling other groups to search the database. Bioinformatics that has facilitated research into metabolic pathways is referred to as chemogenomics. Sometimes when a chemical binds to a target site, it can significantly alter metabolic activity. Massive libraries of chemicals are tested individually on a range of related organisms. For each organism a range of target sites are identified. A range of chemicals which are known to work on those sites are tested. 8.1.A2 Use of databases to identify potential new anti-malarial drugs. http://upload.wikimedia.org/wikipedia/commons/0/02/Mosquito_bite4.jpg

Use of databases to identify potential new anti-malarial drugs. Malaria is a disease caused by the pathogen Plasmodium falciparum. This protozoan uses mosquitoes as a host as well as humans and hence can be passed on by mosquito bites Increasing drug resistance to anti-malarial drugs has lead to the use of bioinformatics and chemogenomics to try and identify new drugs. Fidock et al. article: PREVIEW the article by looking through the article, looking at pictures, and finding the glossary of new words along the sides. Begin reading. As you read: Highlight the words from the glossary when they occur in the text. Underline the topic sentence from each paragraph. Summarize each section (sections have a bold heading) in one or two sentences in your own words without looking back at the text! 8.1.A2 Use of databases to identify potential new anti-malarial drugs. In one study, approx. 300,000 chemicals were screened against a chloroquine-sensitive 3D7 strain and the chloroquine-resistant K1 strain of P. falciparum. Other related and unrelated organisms, including human cell lines, were also screened. (19) new chemicals that inhibit the enzymes normally targeted by anti-malarial drugs were identified Additionally (15) chemicals that bind to malarial proteins were identified – this can help in the location of P. falciparum These results indicate possible new directions for drug research. http://upload.wikimedia.org/wikipedia/commons/0/02/Mosquito_bite4.jpg

The rate of reaction can be calculated using the formula: 8.1.S1 Calculating and plotting rates of reaction from raw experimental results. Remember this lab? 2.5.S2 Experimental investigation of a factor affecting enzyme activity. (Practical 3) Use the results from our classes’ data to calculate the rate of reaction. The rate of reaction can be calculated using the formula:   Rate of reaction (s-1) = 1 / time taken (s) Time taken in enzyme experiments this is commonly the time to reach a measurable end point or when a standard event, caused by the enzyme reaction, has come to pass. This is usually measured by the effects of the accumulation of product, but can as easily be measured by the disappearance of substrates. Enzyme inhibition can be investigated using these two outlines by Science & Plants for Schools: The effect of end product, phosphate, upon the enzyme phosphatase The inhibition of catechol oxidase by lead http://www.scienceexperimentsforkids.us/wp-content/uploads/2011/08/hydrogen-experiments-for-kids-3-img.jpg

Bibliography / Acknowledgments Jason de Nys