Biomolecules, Enzymes and Vitamins PDF

S-BIOL313 Biomolecules Module No. 4. Enzymes and Vitamins MODULE 4 ENZYMES AND VITAMINS Prepared by: JOSEPH L. SAMONTE, Ph.D. Biological Sciences Department College of Science and Computer Studies De La Salle...

S-BIOL313 Biomolecules Module No. 4. Enzymes and Vitamins MODULE 4 ENZYMES AND VITAMINS Prepared by: JOSEPH L. SAMONTE, Ph.D. Biological Sciences Department College of Science and Computer Studies De La Salle University-Dasmariñas Dasmariñas City, Cavite 1  S-BIOL313 Biomolecules Module No. 4. Enzymes and Vitamins TABLE OF CONTENTS Section Title Page 1.0 Gospel Reflection 3 2.0 Module Learning Objectives 4 3.0 Introduction 4 4.0 Enzymes 5 5.0 Vitamins 6 6.0 Vitamins Analysis 8 7.0 Key Take-Aways 9 8.0 References 10 9.0 Enabling Assessment 11 2  S-BIOL313 Biomolecules Module No. 4. Enzymes and Vitamins 1.0 GOSPEL REFLECTION Daniel 1:12 (English Standard Version) 2 “Test your servants for ten days; let us be given vegetables to eat and water to drink. Reflection Point: Using the space below, write a reflective essay based on the gospel above. Relate the bible verse to your personal life and explain how this gospel is connected to this module? ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ 3  S-BIOL313 Biomolecules Module No. 4. Enzymes and Vitamins 2.0 MODULE LEARNING OBJECTIVES At the end of this module, you should be able to: 1. Describe enzymes and their function in chemical reactions. 2. Identify the importance of vitamins as cofactor of enzymes. 3. List the ways in which vitamins are analyzed. 3.0 INTRODUCTION Enzymes are proteins that catalyze body’s chemical reactions. They can increase the rate of reaction as much 106 by reducing the activation energy without affecting the equilibrium. Since the enzymes are proteins, they are made up of amino acids linked together by peptide bonds. The reactant of an enzyme-catalyzed reaction is called as substrate. The specificity of the enzyme for its substrate is known as molecular recognition. Coenzymes are organic molecules commonly derived from vitamins. Vitamin is a substance that body cannot normally synthesize, but they are required in small amount for normal body function. Example of vitamins are niacin, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin H and vitamin K. For the details of the vitamin A, D and E. Motivating Activity (Find the Difference). Find 10 difference between the two pictures below. Figure 1. Fruits and vegetables that contains vitamins (Zakowski 2020). 4  S-BIOL313 Biomolecules Module No. 4. Enzymes and Vitamins 4.0 ENZYMES (NPTEL 2020) Enzymes are generally very specific; they catalyze some specific chemical process; to perform their function, they often need specific substances called co-factors. If the body does not produce this material, it must be present in the diet; these cofactors are called vitamins. Active site of an enzyme is generally a hollow on the protein surface where the substrate can bind. The substrate is generally bound to amino acids present in the binding site by variety of interactions such as van der Waals interaction, H-bonding and ionic bonding. These interactions are to strong enough to hold the substrate for the reaction to take place, but weak enough to allow the product to depart once it is produced. The amino acids present in the active site also assist in the reaction mechanism. For example, nucleophilic amino acid such as serine is commonly involved in enzyme catalyzed reaction mechanisms and will form covalent bond with the substrate as part of the reaction mechanism There are many reasons why enzymes catalyze the reactions. We have seen an example where amino acid in the active site can assist the enzyme mechanism acting as a nucleophile. Another reason why enzyme acts as catalyst is the binding process itself. The active site is not ideal shape for the substrate, but when the binding takes place the shape changes to accommodate the substrate and maximizes the possible interactions of the substrate with amino acids of the active site. This is called as an induced fit. These binding process also force the substrate to adopt a specific conformation which is ideal for the reaction with nucleophile and catalytic amino acids. Furthermore, the binding between the substrate and enzyme is that important bonds in the substrate may be strained and weakened, allowing the reaction takes place more easily. For an example, neurotransmitter acetylcholine is hydrolyzed by the enzyme acetylcholinesterase (Scheme 1). In this process, acetylcholine is bound in the active site such that it is held in position to undergo reaction with nucleophilic serine OH group. A histidine residue is also positioning such a way to act as an acid/base catalyst. H-Bonding between the ester group of the substrate with a tyrosine residue in the active site also aids to weaken the ester linkage, allowing it to be cleaved more easily. Enzyme inhibitors are drugs which inhibit the catalytic activity of enzyme. They can be classified as competitive and noncompetitive inhibitors. Competitive inhibitors compete with the substrate for the active site. The greater the concentration of the inhibitor, there is a less probability for the substrate to enter the active site. On the other hand, the greater the 5  S-BIOL313 Biomolecules Module No. 4. Enzymes and Vitamins concentration of the natural substrate, the less efficient is the inhibition. Noncompetitive inhibitors do not compete with the substrate to enter the active site. Instead, they bind at different region of the enzyme and produces an induced fit, which changes the shape of the enzyme such as the active site no longer available to the substrate. Increasing the concentration of the substrate will have no effect on the level of inhibition. The binding site of the noncompetitive inhibitors is known as allosteric site. It is often present in the enzyme at the start of the biosynthesis. The allosteric site binds the final product of the biosynthesis and provides a feedback control for the biosynthesis pathway. When the concentration of the biosynthesis product is high, the allosteric site is occupied, the activity of the enzyme is off. The final biosynthetic product is thus used as a lead compound for the design of inhibitors that will bind to the allosteric site. The inhibitors can be reversible or nonreversible. In case of reversible inhibitor, the inhibitor binds with the enzyme through intermolecular interactions such as H-bond, ionic bonding and van der Waals interactions. There will be equilibrium between the enzyme- inhibitor complex and free enzyme and unbound inhibitor. The position of the equilibrium will depend on the strength of the intermolecular interactions between the enzyme and inhibitor. On the other hand, in case of non-reversible inhibitors, the inhibitor may react with enzyme generating a covalent bond. Such bonds are strong, and the enzyme remains permanently blocked making the inhibition irreversible. The inhibitor should be selective for the target enzyme to avoid the side effects. There are instances where a enzyme may exist in different forms having different amino acid composition, but identical catalytic reaction. These enzymes are called isozymes. If the variation is in the binding site, it is possible to design drugs that will be the isozyme selective. 5.0 VITAMINS (NPTEL 2020) Coenzymes are organic molecules commonly derived from vitamins. Vitamin is a substance that body cannot normally synthesize, but they are required in small amount for normal body function. Niacin. Nicotinamide adenine dinucleotide (NAD+) is a coenzyme that is used by enzyme as oxidizing agent for biological oxidations. Since niacin is the portion of coenzyme NAD+, it is to be added in the diet so that the body can synthesize NAD+ 6  S-BIOL313 Biomolecules Module No. 4. Enzymes and Vitamins Vitamin B2. Riboflavin (flavin plus ribitol) is known as vitamin B2. It is required to synthesize coenzyme flavin adenine dinucleotide (FAD) which is used as oxidizing agent in enzyme catalyzed oxidation reactions. For example, FAD is used by succinate dehydrogenase to oxidize succinate to fumarate Vitamin B1. Thiamine is known as vitamin B1. It is required to form the coenzyme thiamine pyrophosphate (TPP). TPP is required by enzymes that catalyze the transfer of a two carbon fragment from one species to another. For example, pyruvate decarboxylase enzyme requires TPP for the decarboxylation of pyruvate and transfer the remaining twocarbon fragment to a proton to afford acetaldehyde Vitamin H. Biotin is known as vitamin H, which is synthesized by bacteria the live in intestines. Thus, biotin does not have to be incorporated in diet and the deficiencies are generally rare. Biotin is the coenzyme required by enzymes that catalyze carboxylation of a carbon adjacent to a carbonyl group. For example, acetyl-CoA carboxylate converts acetyl-CoA into malonyl-CoA. HCO3- is used as the source of carboxyl group which is attached to the substrate (Scheme 3). The reaction is also required ATP and Mg2+. Vitamin B6. The coenzyme pyridoxal phosphate (PLP) is derived vitamin B6. The deficiency of vitamin B6 causes anemia. Enzymes that catalyze the reactions of amino acids require PLP. One of the common examples is the transamination using enzyme aminotransferases. Vitamin B12. Enzymes that catalyze certain rearrangement reactions require coenzyme B12 which is derived from vitamin B12. In vitamin B12, cyano group is coordinated to cobalt. In coenzyme B12, this group is replaced by a 5’-deoxyadenosyl group. Coenzyme B12 is used by enzymes which catalyze reactions in that a group (Y) bonded to one carbon changes to hydrogen bonded an adjacent carbon Folic Acid. Tetrahydrofolate (THF) is the coenzyme required by enzymes which catalyze the transfer of a group having a single carbon to their substrates. The one carbon may be methyl (CH3), methylene (CH2) or formyl (CH) group. The coenzyme THF is required for the synthesis of bases in RNA and DNA and synthesis of some aromatic amino acids. Folic acid is the vitamin used for the synthesis of the coenzyme THF. For example, thymidylate synthase is the enzyme that catalyzes the synthesis of T’s from U’s. This transformation requires N5,N10-methylene- THF as a coenzyme. 7  S-BIOL313 Biomolecules Module No. 4. Enzymes and Vitamins Vitamin K. Vitamin K is synthesized by intestinal bacteria and is required for proper clotting of blood. In order for blood to clot, blood-clotting proteins must bind to Ca2+. And since Vitamin K is synthesized by intestinal bacteria its deficiency is rare. Vitamin KH2 is the coenzyme that is derived from vitamin K. The coenzyme vitamin KH2 is required by enzyme that catalyzes the carboxylation of the γ-carbon of glutamate side chains in proteins, forming γ-carboxyglutamates 6.0 VITAMINS ANALYSIS (R-BiopharmAG 2020) Vitamins are essential to our health: Our body needs them for metabolism, for energy production, for the immune system and for the cell formation. A lack of vitamins may cause deficiency symptoms such as circulatory disturbance, metabolic disorder, anemia or muscular atrophy. Thirteen vitamins are regarded as vital for humans:  vitamin A  vitamin B6  vitamin C (ascorbic  vitamin B1  vitamin B7 (biotin) acid)  vitamin B2  folic acid  vitamin D  vitamin B3 (niacin)  vitamin B12  vitamin E  pantothenic acid  vitamin K Since vitamins cannot be produced by the human body itself (with the exception of vitamin D and niacin), they must be consumed with food. A balanced diet is therefore essential. This includes of course fruit and vegetables, but also fish, meat, cereals, nuts and dairy products. Moreover, some foods are artificially enriched with added vitamins. Supplements offered as tablets or powders can contribute to an optimal vitamin supply, too. Especially juices, baby food and dairy products are often advertised to be enriched with vitamins and thereby to contribute to a healthy diet. There are different methods that allow it to examine whether the product actually contains the amount of vitamins that is indicated on the label. For example, immuno- affinity columns (IAC) are one commonly used method in vitamin analysis. These columns are used for sample purification prior to a liquid chromatography using HPLC (high performance liquid chromatography) or LC/MS. However, a more simple and affordable method is an ELISA test. 8  S-BIOL313 Biomolecules Module No. 4. Enzymes and Vitamins Here you can see the different methods for vitamin analysis briefly: Table 1. Methods of vitamins analysis. Method ELISA test Microbiological test Immuno-affinity column Duration Incubation Incubation period: 44-48 2-3 hours for clean-up, period: 25 hours (Pantothenic acid: 24 plus HPLC- or LC/MS- minutes hours) analysis Benefits Easy to use High precision High recovery No toxic Some tests are AOAC-RI Low coefficients of chemicals certified variation required Available Vitamin B12 Vitamin B12 Vitamin B12 for: Folsäure (Folic Folsäure (Folic Acid) Folsäure (Folic Acid) Acid) Biotin Biotin Vitamin B1 Vitamin B2 Vitamin B3 (Niacin) Vitamin B6 Vitamin C Pantothensäure (Pantothenic Acid) Inositol 8.0 KEY TAKEAWAYS Enzymes are generally very specific; they catalyze some specific chemical process; to perform their function, they often need specific substances called co-factors. If the body does not produce this material, it must be present in the diet; these cofactors are called vitamins. Active site of an enzyme is generally a hollow on the protein surface where the substrate can bind. 9  S-BIOL313 Biomolecules Module No. 4. Enzymes and Vitamins Coenzymes are organic molecules commonly derived from vitamins. Vitamin is a substance that body cannot normally synthesize, but they are required in small amount for normal body function. There are different methods that allow it to examine whether the product actually contains the amount of vitamins and this includes ELISA test, Microbiological test and Immunoaffinity column. 8.0 REFERENCES Clark M, Douglas M, Choi J. 2018. Biology 2e. OpenStax: Houston, Texas. [Internet]. [accessed 28 August 2020]. https://openstax.org/books/biology-2e/pages/22-1prokaryotic-diversity National Programme on Technology Enhanced Learning (NPTEL) 2020. Biotechnology Cell Biology Module 13 Enzymes and Vitamins. [Internet]. [accessed 28 August 2020]. https://www.coursehero.com/file/11215583/mod13/ R-BiopharmAG. 2020. Vitamin analysis: New test kits for the determination of vitamin content in food. [Internet]. [accessed 28 August 2020]. https://food.r-biopharm.com/ news/vitamin- analysis-new-test-kits-determination-vitamin-content-food/ The Holy Bible. 2016. English Standard Version. ESV® Text Edition. Copyright © 2001 by Crossway Bibles, a publishing ministry of Good News Publishers. [accessed 28 August 2020]. https://www.biblegateway.com/ Zakowski I. 2020. Vector Stock. Finding differences game cartoon vector image. [accessed 29 August 2020]. https://www.vectorstock.com/royalty-free-vector/finding-differences-game- cartoon-vector-4123306 10  S-BIOL313 Biomolecules Module No. 4. Enzymes and Vitamins 9.0 FORMATIVE ASSESSMENT ASSESSMENT TITLE Proteins ASSESSMENT TYPE No. Est. Duration 60 minutes OUTCOMES 1. Describe enzymes and their function in chemical reactions. ASSESSED 2. Identify the importance of vitamins as cofactor of enzymes. 3. List the ways in which vitamins are analyzed. INSTRUCTIONS: In each of the following multiple-choice questions, place the letter of the correct response in the blank at the left. There is only one correct response for each question. _____1. What is the function of enzymes within living systems? A. structural elements B. neurotransmitters C. catalysts D. hormones _____2. Enzymes have names that A. always end in -ase. B. always end in -in. C. can end in either -ase or -in. D. can end in either -ase or -ogen. _____3. In which of the following is the pairing between enzyme type and enzyme function incorrect? A. lipase – hydrolysis of ester linkages in lipids B. reductase – reduction of a substrate C. kinase – transfer of amino groups between substrates D. dehydrogenase – introduction of a double bond through hydrogen removal _____4. Which of the following is always present in both conjugated enzymes and simple enzymes? A. protein B. a vitamin C. cofactor D. coenzyme _____5. The protein portion of a conjugated enzyme is called a/an A. apoenzyme. B. coenzyme. C. holoenzyme. D. cofactor. 11  S-BIOL313 Biomolecules Module No. 4. Enzymes and Vitamins _____6. An enzyme active site is the location in an enzyme where A. interaction with a substrate occurs. B. interaction with a catalyst occurs. C. catalyst molecules are generated. D. substrate molecules are generated. _____7. Which of the following enzyme properties is explained by the lock-and-key model for enzyme action? A. high turnover rate B. absolute specificity C. susceptibility to denaturation D. susceptibility to deactivation _____8. An enzyme that catalyzes the oxidation of many different alcohols would be described as showing A. absolute specificity. B. stereochemical specificity. C. linkage specificity. D. group specificity. _____9. A plot of enzyme activity (y-axis) vs. pH (x-axis) with other variables constant is a A. straight line with an upward slope. B. line with an upward slope and a long flat top. C. line with an upward slope followed by a downward slope. D. straight horizontal line. _____10. A plot of enzyme activity (y-axis) versus substrate concentration (x-axis) with other variables constant is a A. straight line with an upward slope. B. line with an upward slope and a long flat top. C. line with an upward slope followed by a downward slope. D. straight horizontal line. _____11. Which of the following statements concerning a competitive enzyme inhibitor is correct? A. resembles the substrate in structure B. binds at a site other than the active site C. its effect is never reversible D. its effect can be overcome by decreasing the substrate concentration 12  S-BIOL313 Biomolecules Module No. 4. Enzymes and Vitamins _____12. Which of the following binds to an enzyme at a location other than the active site? A. Substrate B. irreversible inhibitor C. reversible competitive inhibitor D. reversible noncompetitive inhibitor _____13. The number of substrate molecules acted upon per minute by one molecule of enzyme is called the A. active site number. B. enzyme activity number. C. turnover number. D. optimum number. _____14. The final product of a series of enzyme-catalyzed reactions causes the enzyme that catalyzes the first reaction of the series to be inhibited. This is an example of A. positive regulator control. B. substrate control. C. feedback control. D. competitive control. _____15. Which of the following substances is a zymogen? A. peptidase B. pepsin C. procarboxypeptidase D. trypsin _____16. The number of known vitamins is A. 10. B. 13. C. 15. D. 20. _____17. In which of the following pairs of vitamins are both members of the pair water- soluble? A. vitamin A and vitamin C B. vitamin D and vitamin K C. vitamin E and vitamin B12 D. biotin and niacin _____18. Vitamins C and K are important, respectively, in A. bone formation and blood clotting. B. blood clotting and mucous membrane integrity. C. vision and bone formation. D. bone formation and mucous membrane integrity. 13  S-BIOL313 Biomolecules Module No. 4. Enzymes and Vitamins _____19. The major function for B vitamins within the human body is as A. antioxidants. B. components of coenzymes. C. regulators of cell differentiation. D. regulators of calcium ion and phosphate ion concentrations in blood. _____20. All of the B vitamins, except one, contain heterocyclic nitrogen ring systems as part of their structure. The exception is A. riboflavin. B. thiamin. C. pantothenic acid. D. biotin. 14