CHEM 103M Lecture Notes (Finals) PDF

 CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department Preface Congratulations for finishing halfway through your biochemistr...

 CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department Preface Congratulations for finishing halfway through your biochemistry! It wasn’t easy, I know. So give yourself a tap in the back for the job well done. Final term might a bit more challenging in terms of lessons, but I know that you somehow learn now how to deal with the subject from midterms. I am wishing to see improvement in your performance. To guide you in finals, I am providing you the second volume of the lecture notes compilation which includes the topics that are intended to be covered during the Final Term (Weeks 4-6). Concepts, graphics, exercises, and problems are derived from reputable biochemistry textbooks and supplemented with self-written discussion, explanation, and worked examples. While it is not intended to replace the textbooks from which the materials in this compilation are derived, it should serve as a useful reference for you. Exercises, questions and worked problems are placed where appropriate. Enjoy learning! Sir Jelo Page 1 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department Table of Contents Topic Page Unit 5 – Chemistry of Proteins I. Overview 4 A. Definition of Proteins II. Amino Acids A. General Structural Feature of Amino Acids B. Classification of Amino Acids 5 C. Essential Amino Acids D. Amphoteric Properties of Amino Acids III. Peptides A. Formation and General Structural Feature of Peptides 20 B. Nomenclature of Peptides C. Small Peptides of Physiologic Importance IV. Three-Dimensional Structure of Proteins A. Levels of Protein Structure B. Primary Structure of Proteins 25 C. Secondary Structure of Proteins D. Tertiary Structure of Proteins E. Quaternary Structure of Proteins V. Protein Hydrolysis and Denaturation 36 Unit 6 – Chemistry of Enzymes I. General Characteristics of Enzymes A. Structural Features of Enzymes 38 B. Nomenclature of Enzymes C. Six Major Classification of Enzymes II. Enzymatic Activity A. How Enzymes Make Reaction Faster? B. Formation of the Enzyme-Substrate Complex 42 C. Nature of the Active Site D. Factors Affecting Enzyme Activity III. Enzyme Inhibition and Regulation A. Type of Enzyme Inhibition 46 B. Regulatory Mechanisms of Enzymes IV. Role of Enzymes in Medicine A. Enzyme Inhibitors Used as Drugs 51 B. Roles of Enzymes in Disease Diagnosis Unit 7 – Chemistry of Nucleic Acids I. Overview 54 A. Types of Nucleic Acids II. Nucleotides 56 A. Components of Nucleotides III. Structure of Nucleic Acids A. Primary Structure of Nucleic Acids B. Double Helix Structure of DNA 59 C. Higher Order Structure of DNA D. Structural Difference Between DNA and RNA Page 2 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department IV. Central Dogma of Molecular Genetics A. Overview B. Genes 62 C. Replication D. Transcription E. Translation V. Mutations and Viruses A. Types of Mutation 71 B. Repair of Mutation C. Viruses Unit 8 – Metabolism I. Overview A. Nature of Metabolism 76 B. Key Intermediates of Metabolism C. Foods That Are Basis of Human Nutrition II. Biochemical Energy Production A. Stages of Biochemical Energy Production B. Common Catabolic Pathways 83 i. Krebs Cycle ii. Electron Transport Chain and Oxidative Phosphorylation III. Metabolism of Carbohydrates A. Digestion and Absorption of Carbohydrates B. Glycolysis C. Storage Mechanisms and Control in Carbohydrate Metabolism i. Reactions of and conditions that promote: 101  Gluconeogenesis  Glycogenesis and Glycogenolysis ii. Hormonal Controls of Carbohydrate Metabolism iii. Pentose Phosphate Pathway IV. Metabolism of Lipids A. Digestion and Absorption of Lipids B. Catabolism of Glycerol 121 C. β-oxidation of Fatty Acids D. Energy Yield from Oxidation of Fatty Acids E. Ketone Bodies V. Metabolism of Proteins A. Digestion and Absorption of Proteins B. Amino Acid Utilization C. Catabolism of the Nitrogen Portion of Amino Acids 132 i. Transamination ii. Oxidative Deamination iii. Urea Cycle D. Fates of the Amino Acid Carbon Skeleton CONCLUSION 141 Page 3 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department Unit 5 – CHEMISTRY OF PROTEINS INTENDED LEARNING OUTCOMES At the end of this unit, you will be able to: 1. Categorize amino acids based on functional group and polarity; 2. Explain the amphoteric property of amino acids owing to its amino and carboxyl groups; 3. Illustrate the formation of peptides as a reaction between different amino acids; 4. Determine the sequence of amino acids and name of peptides; 5. Explain the importance of some small peptides; 6. Describe the different levels of protein structure and their interrelationship with one another; 7. Relate protein structure to its physiological function; and 8. Differentiate hydrolysis and denaturation of proteins. UNIT OUTLINE Topic Page I. Overview 4 II. Amino Acids A. General Structural Feature of Amino Acids B. Classification of Amino Acids 5 C. Essential Amino Acids D. Amphoteric Properties of Amino Acids III. Peptides A. Formation and General Structural Feature of Peptides 20 B. Nomenclature of Peptides C. Small Peptides of Physiologic Importance IV. Three-Dimensional Structure of Proteins A. Levels of Protein Structure B. Primary Structure of Proteins 25 C. Secondary Structure of Proteins D. Tertiary Structure of Proteins E. Quaternary Structure of Proteins V. Protein Hydrolysis and Denaturation 36 I. OVERVIEW When scientists began studying nutrition in the early 19 th century, they quickly discovered that natural products containing nitrogen were very essential for the survival of animals. This class of compound was then coined as protein by the Swedish chemist Jacob Berzelius in 1939 which was derived from the Greek word “proteios” which means of first importance. Proteins are the most important macromolecule in the body because it plats a lot of important physiological functions. It accounts for 15% of total cell’s mass and for almost 50% of its dry weight. Page 4 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department What makes a protein a protein?  Proteins are naturally occurring unbranched polymers (long chain) of amino acids connected together by peptide bonds.  FUN FACT: When proteins were first discovered, physiological chemists (the term biochemist was not yet used before) did not realize that proteins were made up of smaller units, amino acids, although the first amino acid has already been isolated in 1830. In fact, they believe that proteins were incorporated whole into tissues of animal. This misconception was laid to rest when the process of digestion came to light. It became clear that ingested proteins were broken down into smaller compounds. II. AMINO ACIDS A. General Structural Feature of Amino Acids An amino acid is a compound containing a carboxyl (-COOH) group and amino (-NH2) group at the same time. There are more than 300 naturally occurring amino acids, but only 20 of them are found in humans. These are known as the Standard Amino Acids (SAA). These Standard Amino Acids are α-amino acids because they have a carboxyl group and a primary amino group attached in the same carbon, known as the α-carbon (See Figure below). The sole exception is proline, as it has secondary amino group, although for uniformity we will also refer proline as an α-amino acid. COOH Carboxyl group Amino group H2 N C H R R Side Chain The 20 Standard Amino Acids differ in the structure of their side chains, and, therefore, distinguishes them from each other. Enantiomers of Standard Amino Acids If you noticed in the general structure of the Standard Amino Acids, there are four different group around the α-carbon. This means that the α-carbons of the Standard Amino Acids are stereogenic centers, except glycine since the side chain of glycine is H resulting to 2 identical hydrogens in its α-carbon (See Figure on the right). Because 19 of the Standard Amino Acids have stereogenic centers, they also exist as enantiomers – D or L. Page 5 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department To represent the D and L enantiomers of the Standard Amino Acids, we use the Fischer projection like in monosaccharides. The Fischer projection of the D and L Standard Amino Acids is illustrated below: The location of the amino group (-NH2) of the α-carbon of a Standard Amino Acid in its Fischer projection determines its type of enantiomer. For L amino acids, the amino group of the α-carbon is directed to the left. For D amino acids, the amino group of the α-carbon is directed to the right. In nature and in proteins, the Standard Amino Acids are L isomers. B. Classification of Standard Amino Acids Because the standard amino acids are distinguished from each other by their side chains, we can classify them according to the characteristics of their side chains. The most common way is based on polarity of side chains. Thus, standard amino acids can either be polar or non-polar. Polar amino acids can be further categorized as polar neutral, polar acidic, or polar basic. The table below shows the differences among the different classes of amino acids. Polar Properties Non-Polar Neutral Acidic Basic monoamino monoamino monoamino diamino monocarboxylic monocarboxylic Structure dicarboxylic monocarboxylic acid with non- acid with polar acid acid polar side chain side chain Has negative Has positive charge near charge near Do not ionize pH 7 due to Chemical pH 7 due to the at near pH 7 the Characteristic Hydrophobic protonation of (physiological deprotonation of Side Chain the amino pH). of the carboxyl group in the group in the side chain. side chain. Page 6 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department The standard amino acids are referred by their common names. These names are often abbreviated as three-letter code or one-letter code. The illustration on the next page displays the names and complete structures of the 20 standard amino acids. NON-POLAR AMINO ACIDS Page 7 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department POLAR NEUTRAL AMINO ACIDS POLAR BASIC Page 8 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department POLAR ACIDIC AMINO ACIDS C. Essential Amino Acids Essential amino acids are Standard Amino Acids that the human body cannot adequately synthesize and must, therefore, be obtained from dietary sources. There are 10 essential amino acids necessary for normal growth of a child. These amino acids can be easily memorized easily using the mnemonic PVT. TIM HALL which stands for: Phenylalanine, Valine, Threonine, Tryptophan, Isoleucine, Methionine, Histidine, Arginine, Leucine, and Lysine. Arginine is only essential for infants for normal growth, but becomes nonessential amino acid as they grow into adulthood. Infants that are born prematurely cannot make sufficient quantities of some nonessential amino acids and these amino acids become conditionally essential amino acids until the baby matures. In this situation the conditionally essential amino acids must be obtained through diet. The human milk and infant formula milk contain adequate amounts of these conditionally essential amino acids. Page 9 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department A complete dietary protein (high-quality protein) is a protein that contains all of the essential amino acids in adequate amounts as the body needs them. Proteins from animal sources are usually complete dietary protein. Example, casein from milk and albumin from eggs are considered complete dietary proteins. An incomplete dietary protein is a protein that does not contain in adequate amounts, relative to the body’s needs, of one or more of the essential amino acids. The essential amino acid that is missing or present in an inadequate amount in a incomplete dietary protein is known as a limiting amino acid. Gelatin is an incomplete dietary protein having tryptophan as its limiting amino acid. Protein from plant sources are generally incomplete dietary proteins, with three common limiting amino acids lysine, methionine, and tryptophan. Soy protein is the only common plant protein that is considered complete dietary protein. However, mix of plant proteins generally provide complete dietary protein, such as in the case of rice and beans. Proteins from rice and beans when eaten together are known complementary dietary protein. D. Amphoteric Properties of Standard Amino Acids Recall that an amphoteric substance is a substance that can act as an acid in basic environment and can act as a base in an acidic environment. Amino acids exhibit this amphoterism. How is amphoterism possible in amino acids? The Standard Amino Acids have both carboxyl group (-COOH) and amino group (-NH2) in one molecule. We learned in organic chemistry that –COOH is an acidic group while –NH2 is a basic group. H Basic Acidic group H2 N C COOH group R R At pH near neutral, carboxyl group (-COOH) has the tendency to lose a proton (H+), producing a negatively charged conjugate base known as carboxylate. RCOOH  RCOO- + H+ carboxylate ion In the same manner, amino group (-NH2) have the tendency to accept a proton (H+), producing a positively charged conjugate acid known as quaternary ammonium ion. R – NH2 + H+  R – NH3+ quaternary ammonium ion Page 10 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department Because of this acid-base property of the carboxyl and amino groups, in aqueous solutions, the –COOH of the Standard Amino Acids is deprotonated while the –NH2 is protonated. We can characterize this as an intramolecular acid-base reaction. When this happens, the Standard Amino Acid assumes the structure: H Quaternary ammonium H3N+ C COO- carboxylate R R This structure is known as the zwitterion form of a Standard Amino Acid. A zwitterion or dipolar ion is a molecule having a positive charge on one side and negative charge on the other. Take note that while a zwitterion has charged groups it is neutral molecule because the number of groups with positive charge is equal to the number of groups with negative charge resulting to zero net charge. In solid state, the standard amino acids exist as zwitterions. Structural Changes of the Zwitterion at Varying pH The zwitterion structure of a Standard Amino Acid changes when the pH of the solution containing the Standard Amino Acid is changed from neutral to either acidic (low pH) or basic (high pH). When the solution is made acidic, the H+ concentration is increased by adding an acid, such as HCl. When the solution of a Standard Amino Acid is made acidic (abundant H +), the carboxylate portion of the zwitterion is protonated (accepts H+) to form a positively charged species. H H I I H3N – C – COO_ + + H+ + H3N – C – COOH I I R R Zwitterion Positively charged species Page 11 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department When the solution is made basic, the OH- concentration is increased by adding a base, such as NaOH. When the solution of a Standard Amino Acid is made basic (abundant OH -), the quaternary ammonium portion of the zwitterion is deprotonated (loses H+) to form a negatively charged species. H H I I H3N+ – C – COO_ + OH- H2N – C – COO- I I R R Zwitterion Negatively charged species Thus in aqueous solutions, 3 different form of Standard Amino Acid forms can exist – zwitterion, negative ion, and positive ion. These forms are in equilibrium with each other, and the dominant form is determined by the pH of the solution. The equilibrium can be represented as follows: H H H I OH- I OH- I + H3N – C – COOH H3N – C – COO_ + H2N – C – COO- I I I H+ H+ R R R Acidic solution zwitterion Basic solution (low pH) (high pH) positive ion form negative ion form For example, observe the structural changes in the ionization of alanine below: H H H I OH- I OH- I H3N+ – C – COOH H3N – C – COO_ + H2N – C – COO- I I I CH3 H+ H+ CH3 CH3 Acidic solution zwitterion Basic solution (low pH) (high pH) positive ion form negative ion form So far, we assume that the side chain (R group) of a Standard Amino Acid remains unchanged in solution as the pH is varied. That is only the case for non-polar amino acids and some polar neutral amino acids but not for the acidic or basic ones. For acidic and basic amino acids, the side chain can also acquire a charge because it contains an amino group or a carboxyl group that can, respectively, gain or loses a proton as the pH of the solution is varied. Because of the extra site that can be protonated or deprotonated, acidic and basic amino acids have four charged forms in the solution. Page 12 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department Acidic Amino Acids in Aqueous Solution To illustrate the different forms of acidic amino acids in aqueous solution, let us study the ionization of aspartic acid below:  Notice that the zwitterion for aspartic acid is predominant in a moderately acidic solution. That is the characteristic of acidic amino acids.  Notice as well that the carboxyl group of the α-carbon was first to deprotonate than the carboxyl group in the side chain. This is due to the fact that the carboxyl group of the α- carbon is a much stronger acid than the carboxyl group in the side chain. Basic Amino Acids in Aqueous Solution To illustrate the different forms of basic amino acids in aqueous solution, let us study the ionization of lysine below:  Notice that the zwitterion for lysine is predominant in a moderately basic solution. That is the characteristic of basic amino acids.  Notice as well that the quaternary ammonium group of the α-carbon was first to deprotonate than the quaternary ammonium group in the side chain. This is due to the fact that the amino group of the α-carbon is a much weaker base than the amino group in the side chain. Page 13 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department The ionization in the previous page is only true for lysine and arginine. Histidine has a different ionization property as shown below.  As you see in the illustration, the quaternary ammonium group of the side chain was first to deprotonate than the quaternary ammonium group in the α-carbon. This is due to the fact that the imidazole is a much weaker base than the amino group in the α-carbon. pKa Values of the Ionizable Groups of Standard Amino Acids Form the previous discussions, the –COOH and –NH2 groups of the Standard Amino Acids, whether in α-carbon and or in side chain, can be ionized depending on the pH of the solution. These are known as ionizable groups. Each ionizable group in a Standard Amino Acid is associated with a specific pKa value that is experimentally determined. Listed below is the pKa value of the ionizable groups in Standard Amino Acids. Page 14 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department Most of the Standard Amino Acids have only 2 pKa values corresponding to its 2 ionizable groups, one for the α-carboxyl group and one for the α-amino group. There are other amino acids with 3 pKa values. This is when the side chain can appreciably ionize, such as in the case of acidic and basic amino acids. What is the significance of the pKa values? Let us take the ionization of alanine below as an example to show the significance of the pKa values. The pKa values of the different ionizable groups in alanine are already assigned. Each pKa value corresponds to the pH at which the concentrations of the protonated and unprotonated forms are equal. This means that:  at pH 2.4 the concentration of positive ion of alanine is equal to the concentration of its zwitterion. At pH lower than 2.4 the positive ion predominates, and at pH above 2.4 the zwitterion predominates.  at pH 9.9 the concentration of the zwitterion of alanine is equal to the concentration of its negative ion. At pH lower than 9.9 the zwitterion predominates, and at pH above 9.9, the negative ion predominates. Let us take ionization of aspartic acid as another example: The equilibrium above showing ionization of aspartic acid means that:  at pH 2.0 the concentration of positive ion of aspartic acid is equal to the concentration of its zwitterion. At pH lower than 2.0 the positive ion predominates, and at pH above 2.0 the zwitterion predominates.  at pH 3.9 the concentration of the zwitterion of aspartic acid is equal to the concentration of its intermediate pH form. At pH lower than 3.9 the zwitterion predominates, and at pH above 3.9 the intermediate pH form predominates.  at pH 9.9 the concentration of the intermediate pH form of aspartic acid is equal to the concentration of its negative ion. At pH lower than 9.9 the intermediate pH form predominates, and at pH above 3.9 the negative ion predominates. Page 15 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department Isoelectric Point An important pH value, relative to the various forms an amino acid can have in solution, is the pH of at which it exists primarily in its zwitterion form. This pH is known as the isoelectric point given the symbol pI. At isoelectric point, almost all amino acid molecules in a solution (> 99%) exist as zwitterions. Experimentally, the isoelectric point is determined by plotting a titration curve for each amino acid. Since, the pKa values of the ionizable groups of all the Standard Amino Acids are already determined, it would be very easy to calculate for its pI. To calculate the isoelectric point, we use the formula: 𝑝𝐾𝑎1 + 𝑝𝐾𝑎2 𝑝𝐼 = 2 Where: pKa1 and pKa2 are the pKa values besides the zwitterion. For example, let us calculate the isoelectric point of aspartic acid given its ionization below: Solution:  The pKa values between the zwitterion of aspartic acid is 2.09 and 3.86; therefore, the pI of aspartic acid is: 2.0 + 3.9 𝑝𝐼 = = 2.95 2  This means that at pH 2.95, aspartic acid exists primarily as a zwitterion. Separation of Amino Acids by Electrophoresis (Application of Isoelectric Point) Electrophoresis is a common method for separating charged molecules in an electric field. In paper electrophoresis, paper is used as a matrix where separation take place. In gel electrophoresis, cross-linked gelatin-like substance is used as matrix. For the purpose of our discussion, we will use paper electrophoresis. The procedure of paper electrophoresis is summarized below: 1. A small amount of sample solution containing amino acids is placed in the center of a solid matrix. Both ends of the matrix is soaked in ionic buffer solution having a specific pH. 2. An electric voltage is applied at the electrodes immersed in the buffer. Cathode refers to the negative electrode, while anode refers to the positive electrode. The amino acids in the sample will then migrate to the electrodes, and the direction of migration is determined by its form in the buffer Page 16 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department A simplified paper electrophoresis set up is shown below: Cathode Anode Matrix Sample Buffer As the electric voltage is applied amino acids in their positive ion form will migrate to the cathode (negative electrode), while amino acids in their negative ion form will migrate to the anode (positive electrode). There will be no migration if the amino acid is in its zwitterion form. It would stay in the original location of the sample. Predicting the Migration of Amino Acids During Electrophoresis The pH of the buffer used during electrophoresis would determine what form would an amino acid be, whether it would be in its positive ion form, negative ion form, or zwitterion form. We can predict at which electrode will an amino acid migrate by knowing its form when subjected to the buffer. Example: 1. Predict the migration of alanine during electrophoresis when using buffer having a pH of 10. |I|I|I - + pH 10 Alanine Cathode Anode Page 17 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department Solution: 1. Recall the ionization of alanine in the previous discussion. The isoelectric point of alanine is around 6.2. 2. The pH of the buffer in the experiment is pH 10. At this pH, the negative ion form of alanine predominates. Thus, it will migrate to the direction of the anode (positive electrode). Result of the electrophoresis: |I|I|I - + Alanine Cathode Anode (Note that the color of the spot in the display above is only a convenient reference, since these amino acids are colorless. To visualize the location of the amino acids after electrophoresis, ninhydrin solution is sprayed on the matrix. A violet coloration will appear if ever an amino acid is present in the area, such as shown below.) Page 18 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department 2. A sample contains a mixture of alanine (pI=6.2), arginine (pI=10.8), isoleucine (pI=6.05), and aspartic acid (pI=2.95). Predict the migration of the amino acids in the sample during electrophoresis using a pH 6 buffer. |I|I|I - + Cathode pH=6 Anode (alanine, arginine, isoleucine, aspartic acid) Solution: 1. At pH 6 alanine and isoleucine exists as zwitterions; therefore, they will not migrate in the matrix. 2. At pH 6, the positive ion form of arginine predominates; therefore, it will migrate to the cathode. 3. At pH 6, the negative form of aspartic acid predominates; therefore, it will migrate to the anode. Result of the electrophoresis: |I|I|I - + Ala Arg Ile Asp Cathode Anode Page 19 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department III. PEPTIDES A. Formation and General Structural Feature of Peptides A peptide is a chain of amino acids that is formed when the carboxyl group of the α-carbon of an amino acid reacts with the amino group of the α-carbon of the second amino acid. The bond formed in this reaction is generally an amide bond. However, for peptides proteins, the bond is called a peptide bond. The formation of peptide is clearly a type of dehydration reaction, since water is a product after the reaction. For every peptide bond that is formed in a reaction, there is also a production of 1 water molecule. Classification of Peptides - Peptides are classified according to the number of amino acid residues present. For example, the peptide below is considered a dipeptide because it contains 2 amino acid residues: Tripeptide is the classification of peptides with 3 amino acid residues, tetrapeptide for those with 4 amino acids, pentapeptide for those with 5 amino acids, and so on. Normally, when a peptide has 10-20 amino acid residues it is already referred as an oligopeptide. When it contains more than 20 amino acid residues already it is already referred to as polypeptide. NOTE:  Peptides are not proteins. However, proteins are composed of polypeptides. As we will later find out, some proteins may contain more than one polypeptide chain and other non-amino acid group. Page 20 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department General Structural Feature of a Peptide Chain Let us study the structure of a tetrapeptide below to describe the structural features of a peptide: A peptide chain has directionality because its ends are different – an α-amino group is present at one end, and an α-carboxyl group at the other. The amino acid residue with a free α-amino group is the N-terminal residue; the residue at the other end, which has a free α-carboxyl group, is the C-terminal residue. Thus, in the example above, alanine is the N-terminal amino acid, while serine is the C-terminal amino acid. By convention, the sequence of amino acids in a polypeptide chain is written starting with the N-terminal residue. The peptide backbone of a peptide or a protein consists of the repeated sequence –N–Cα– C–, where N stands for the amino or amide nitrogen, the Cα is α-carbon atom of an amino acid residue, and the final C is the carbonyl carbon of the amino acid, which in turn is linked to the amide N of the next amino acid down the line. The peptide backbone is rich in hydrogen-bonding potential. Each residue contains a carbonyl group (C=O) which is a good hydrogen bond acceptor, and, with the exception of proline, an NH group, which is a good hydrogen bond donor. These groups will interact with the groups in the side chains of some amino acid residues stabilizing the overall structure of the protein. Acid-Base Properties of Peptides Peptides contain only one free α-amino group and one free α-carboxyl group, at opposite ends of the chain. These groups can ionize as they do in free amino acids, although the pKa values are different because an oppositely charged group is no longer linked to the carbon. The α-amino and α-carboxyl groups of all nonterminal amino acids are covalently joined in the peptide bonds, which do not ionize and thus do not contribute to the total acid-base behavior of peptides. However, the side chain (R groups) of some amino acids can ionize, and in a peptide these contribute to the overall acid-base properties of the molecule. Thus the acid-base behavior of a peptide can be predicted from its free α-amino and α-carboxyl groups as well as the nature and number of its ionizable side chains. Like free amino acids, peptides have characteristic isoelectric point (pI) at which they do not move in an electric field. At its isoelectric point, the peptide has equal number of positively Page 21 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department and negatively charged groups. These properties are exploited in some of the techniques used to separate peptides and proteins. As an example, consider the peptide below. Its structure reveal that it has -1 net charge due to the ionization of the side chain at the aspartic acid residue. Thus, this structure of the peptide is not in an environment with pH equal to its isoelectric point, Net charge = +1 (free amino) + (-1) (free carboxyl) + (-1) (aspartic acid side chain) = -1 On the other hand, the peptide below has a zero net charge, since the aspartic acid residue was not ionized. This is the form of the peptide when it is subjected in a pH equal to its isoelectric point. Net charge = +1 (free amino) + (-1) (free carboxyl) =0 It should be emphasized that the pKa value for an ionizable side can change somewhat when an amino acid becomes a residue in a peptide. The loss of charge in the α-carboxyl and α-amino groups, the interactions with other peptide side chain, and other environmental factors can affect the pKa. The pKa values for side chain given previously can be a useful guide to the pH range in which a given group will ionize, but they cannot be strictly applied to peptides. B. Nomenclature of Peptides Peptides are named using the following rules set by IUPAC: 1. The C-terminal amino acid residue keeps its full name. 2. All other amino acids have names that end in –yl. The –yl suffix replaces the -ine or –ic acid ending of the amino acid name, except for tryptophan (tryptophyl), cysteine (cysteinyl), glutamine (glutaminyl), and asparagine (asparaginyl). 3. The amino acid naming sequence begins at the N-terminal acid residue. Page 22 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department Examples: H O H H O H H O H H O I II I I II I I II I I II H3 N+ – C – C – N – C – C – N – C – C – N – C – C – O- I I I I CH3 CH2 CH2 CH2 I I I SH OH Alanine Cysteine Phenylalanine Serine Name: alanylcysteinylphenylalanylserine Abbreviations: Ala-Cys-Phe-Ser or ACFS H O H H O H H O H H O I II I I II I I II I I II H3 N – C – C – N – C – C – N – C – C – N – C – C – O- + I I I I CH3 CH2 CH2 CH2 I I I SH SH COOH Alanine Cysteine Cysteine Aspartic acid Name: alanylcysteinylcysteinylaspartic acid Abbreviations: Ala-Cys-Cys-Asp or ACCD Page 23 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department Isomeric Peptides Given 20 different amino acids, there are countless possible combination of peptides. The number of possible of isomers in a peptide is given by 20n, where n is the number of amino acid residues. Peptides that contain the same kind and number of amino acids but differ in order are called isomeric peptides. - Example: H O H H O H O H H O I II I I II I II I I II H3 N+ – C – C – N – C –C – O- H3 N+ – C – C – N – C –C – O- I I I I CH3 H H CH3 Alanylglycine Glycyalanine C. Small Peptides of Physiological Importance 1. Oxytocin and Vasopressin These are peptide hormones that are both produced in the pituitary gland. Each hormone is a nonapeptide, with six of the amino acid residues held in the form of a loop by a disulfide bond formed from the interaction of 2 cysteine residues. Structurally, these nanopeptides differ in the amino acid present in positions 3 and 8 of the peptide chain. In both structures, an amino group replaces the C-terminal single-bonded oxygen atom. Oxytocin regulates uterine contraction and lactation. Vasopressin, also called as antidiuretic hormone (ADH), regulates the excretion of water in kidneys and affects blood pressure. 2. Enkephalins These are pentapeptide neurotransmitters produced by the brain itself that bind at receptor sites in the brain to reduce pain. The two well–known enkephalins are Met-enkephalin and Leu-enkephalin (sequence shown below), whose structures differ only at the C-terminal end of the peptide; this amino acid difference is incorporated in their names. Met-enkephalin: Tyr-Gly-Gly-Phe-Met Leu-enkephalin: Tyr-Gly-Gly-Phe-Leu Page 24 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department The painkillers morphine and codeine can bind at the same receptor sites in the brain as the naturally occurring enkephalins, and thus can reduce pain. The pain relief of morphine and codeine lasts long than enkephalins because they are not affected by the enzymes in the brain that normally hydrolyzes the peptide bonds in enkephalins. 3. Glutathione This is a tripeptide with the sequence Glu-Cys-Gly which is present in significant concentrations in most cells which serves as an antioxidant, protecting cellular component against oxidizing agents, such as peroxides and superoxides (highly reactive species of oxygen often generated within the cell in response to bacterial invasion. The structure of glutathione is a bit odd. The glutamic acid is bonded to cysteine through the side-chain carboxyl group rather than through its α-carbon carboxyl group. H O H H O H H O I II I I II I I II H3 N+ – C – CH2 – CH2 –C – N – C – C – N – C –C – O- I I I COOH CH2 H carboxyl of the I α-carbon SH IV. THREE-DIMENSIONAL STRUCTURE OF PROTEINS The term protein is reserved for polypeptides with a large number of amino acid residues, usually more than 40 residues. A protein may contain only one or more than one polypeptide chain. For this reason, proteins can be classified as: 1. Monomeric proteins – contain only one polypeptide chain (protein subunit) 2. Multimeric proteins – contain more than one protein subunits. The protein subunits in a multimeric protein may be all identical to each other or completely different from each other. An example of a multimeric protein is insulin, composed of 2 protein subunits that are different from each other. Proteins may also contain non-amino acid groups known as prosthetic groups. Thus, proteins can also be classified as: 1. Simple proteins – only amino acid is present 2. Conjugated proteins – contain amino acids and prosthetic groups Types of Conjugated Proteins: Class Prosthetic Group Examples Hemoproteins heme hemoglobin and myoglobin Lipoproteins lipid HDL and LDL Glycoproteins carbohydrates gamma-globulin and mucin Phosphoproteins phosphate casein Nucleoproteins nucleic acid ribosomes Metalloproteins metal ferritin Page 25 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department A. Levels of Protein Three-Dimensional Structure Due to the enormous size of a protein, it can assume many different conformations (three- dimensional structure). Of these many structures, one or a few have biological functions. These are called native conformations. The native conformation of a protein has no predictable repeating structure, but it is not random. Meaning to say, although there are no specific rules for determining how a protein would look, the same native conformation is found in all molecules of a given protein. Describing a protein’s overall three- dimensional structure is complex; thus, we define it in terms of four levels of structure – primary, secondary, tertiary, and quaternary. Primary structure of a protein is the order at which the amino acid residues are covalently linked together. For example, Leu-Gly-Thr-Val-Arg-Asp-His has a different primary structure from the peptide Val-His-Asp-Leu-Gly-Arg-Thr, even though both have the same number and kinds of amino acids. Secondary structure of a protein is the localized arrangement of the atoms in the peptide backbone. This arrangement of the peptide backbone is maintained by hydrogen bonds. There are a lot of types of secondary structure of proteins that will later examine in this lecture notes. Tertiary structure of a protein is the over-all three-dimensional arrangements of all the atoms im a polypeptide chain, including the side-chains and prosthetic groups. Several covalent and non- covalent forces are responsible for maintaining a protein’s tertiary structure. As mentioned previously, a protein may consist multiple polypeptide chains called subunits. Quaternary structure is the arrangement of these subunits with respect to one another. It is noteworthy that quaternary structure exists only for those proteins having multiple subunits (multimeric proteins). The quaternary structure of a protein is mediated by non-covalent interactions. All proteins have until tertiary structure, but not all proteins have quaternary structures. B. Primary Structure of a Protein Page 26 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department The amino acid sequence of a protein, its primary structure, determines its three-dimensional structure, which, in turn, determine its functions. The sequence of amino acids in a protein’s primary structure is decided by the genes. What is the importance of knowing the primary structure of a protein? A change in amino acid sequence of a protein may or may not matter, depending on what kind of change it is. Let us consider 2 cases below to describe and explain the effects of changing a protein’s primary structure: 1. Animal Insulin as Substitute for Human Insulin Insulin is necessary for proper utilization of carbohydrates. Human insulin consists of two chains having a total of 51 amino acids. The two chains are connected by disulfide bonds (See figure below). People with severe diabetes must take insulin injections. The amount of human insulin available is far too small to meet the need for it, so bovine insulin (from cattle) or insulin from hogs or sheep is used instead. Insulin from these sources is similar, but not identical, to human insulin. The differences are entirely in the 8, 9, and 10 positions of the A chain and the C-terminal position of the B chain (See the table above). The remainder of the molecule is the same in all four varieties of insulin. Despite the slight differences in structure, all of these insulins perform the same function and even can be used by humans. However, none of the other three is quite as effective in humans as human insulin. Another factor showing the effect of substituting one amino acid for another is that sometimes patients become allergic to, say, bovine insulin but can switch to hog or sheep insulin without experiencing an allergic reaction. 2. Sickle Cell-Anemia In contrast to the previous example, some small changes in amino acid sequence make a great deal of difference. One of the most striking demonstrations for this is found in the hemoglobin associated with sickle-cell anemia. Page 27 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department Human hemoglobin is composed of four subunits, 2 α chains and 2 β chains, which sums up to a total of 574 amino acid residues (See Figure below). Some people develop a genetic defect resulting to a slightly different kind of hemoglobin in their blood, called HbS. This hemoglobin differs from the normal type only in the β chains and only in one position on these 2 chains: The glutamic acid in the sixth position of normal HB is replaced by a valine residue in HbS. This change affects only a single amino acid residue in a molecule containing 574 amino acid residues, yet it is enough to produce a very serious disease, sickle cell anemia. The name is based from the fact that red blood cells containing HbS assume a sickle shape (See figure on the next page). The sickled cells tend to become trapped in small blood vessels, cutting off circulation and thereby causing organ damage. Normal red blood cells Red blood cells with HbS Page 28 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department C. Secondary Structure of a Protein The next level of protein structure is the secondary structure, which refers to regular localized arrangement of polypeptide backbone of the protein. Recall, that the backbone just refers to the polypeptide chain apart from the side chain – so all we mean here is that secondary structure does not involve R group atoms. The secondary structure is stabilized by hydrogen bond between the carbonyl group (C=O) and the N-H group in the peptide backbone. There are two commonly occurring secondary structures in proteins – the α helix and the β pleated sheet. These 2 are periodic structures, meaning their features repeat in regular intervals. α Helix - The α helix is helical (spiral) repeating structure of a polypeptide chain. The shape of the helix is maintained by hydrogen bond between the carbonyl (–C=O) of one amino acid and an N-H of another amino acid located four residues further along the polypeptide chain, that is between the C=O of the 1st amino acid and the N-H of the 5th amino acid along the chain. In turn, one turn of the helix comprises 3.6 amino acid residues (3 N-Cα-C, 1 C=O, and 1 N-H). In an α helix, all side chains extend outward of the spiral. The linear distance between corresponding points on successive turns, called the pitch, is 5.4 Å (0.54 nm). The helical conformation is very stable because it allows for a linear arrangement of the atoms involved in the hydrogen bonds, which gives the bonds maximum strength. The hydrogen bonds are parallel to the direction of the polypeptide. The following are factors that can disrupt the α helix: 1. Presence of the amino acid proline Proline is known as a helix breaker. It creates a bend in the backbone once it its incorporated in a peptide, thus it destabilizes the helical conformation. 2. Presence of proximate similarly charged groups Amino acids with similarly charged side chains that are near to each other cause electrostatic repulsion destabilizing the helical conformation. Thus, there is a disruption in the α helix when lysine and arginine are too close to each other in a polypeptide because these amino acids both have positively charged side chains and would repel from each other. This is also true for glutamic acid and aspartic acid. Page 29 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department 3. Presence of bulky groups Whenever amino acids with bulky side chains are close to each other in a polypeptide chain, it can cause crowding which results in steric repulsion. Steric repulsion also weakens the stability of the helical conformation. The α helical content of proteins ranges widely, from essentially none to almost 100%. β Pleated Sheet In this type of secondary structure, the peptide backbone is almost fully extended. The hydrogen bonds can be formed between different parts of a single chain that is doubled back on itself (intrachain bonds) or between different chains (interchain bonds). Adjacent strands in a β sheet can run in opposite directions (antiparallel β sheet) or in the same direction (parallel β sheet). In the antiparallel arrangement, the NH group and the C=O group of each amino acid are respectively hydrogen bonded to the CO group and the NH group of a partner on the adjacent chain. Page 30 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department In the parallel arrangement, the hydrogen-bonding scheme is slightly more complicated. For each amino acid, the NH group is hydrogen bonded to the CO group of one amino acid on the adjacent strand, whereas the CO group is hydrogen bonded to the NH group on the amino acid two residues farther along the chain. Turns and Loops Reversals in the direction of polypeptide chains occur in most proteins. Many of these reversals are accomplished by a common structural element called the reverse turn (aka the β turn or hairpin turn). In many reverse turns, the C=O group of residue (i) of a polypeptide is hydrogen bonded to the N-H group of residue i+3. This interaction stabilizes abrupt changes in direction of the polypeptide chain. In other cases, more elaborate structures are responsible for chain reversals. These structures are called loops or sometimes Ω loops (omega loops) to suggest their overall shape. These loops do not have regular periodic structures unlike α helices and β sheets. Nonetheless, loop structures are often rigid and well define. Turns and loops invariably lie on the surfaces of proteins and thus often participate in interactions between proteins and other molecules. Page 31 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department D. Tertiary Structure of a Protein The tertiary structure is the over-all three-dimensional arrangement of all the atoms in a protein. This includes the conformations of the side chains and the positions of any prosthetic groups. Forces that Stabilize Tertiary Structure The illustration below shows different interactions that can stabilize a protein’s tertiary structure. 1. Disulfide bond Recall, the 2 cysteine residues can react to form the dimer cystine (disulfide bond). When a cysteine residue is in one chain and another cysteine residue is in another chain (or in another part of the same chain), formation of a disulfide bond provides a covalent linkage that binds together the two chains or the two parts of the same chain. Page 32 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department 2. Side-chain hydrogen bonding This interaction can occur between polar groups on side chains or between side chains and the peptide backbone. 3. Electrostatic Attraction (Salt Bridge) This can occur between 2 amino acid residues with ionized side chains – that is, between an acidic amino acid (-COO-) and a basic amino acid (–NH3+). The two are held together by simple ion-ion attraction. 4. Hydrophobic Interaction In aqueous solution, globular proteins usually turn their polar groups outward, toward the aqueous solvent, and their nonpolar groups inward, away from the water molecules. The nonpolar groups prefer to interact with each other, excluding water from these regions. The result is a series of hydrophobic interactions. Although this type of interaction is weaker than hydrogen bonding or salt bridges, it usually acts over large surface areas, so that the interactions are collectively strong enough to stabilize a loop or some other tertiary structure formation. 5. Metal Ion Coordination Two side chains with the same charge would normally repel each other, but they can also be linked via a metal ion. For example, two glutamic acid side chains (-COO-) would both be attracted to a magnesium ion (Mg 2+), forming a bridge. This is one reason that the human body requires certain trace minerals—they are necessary components of proteins. The three-dimensional conformation of a protein is the result of the interplay of all the stabilizing forces. Not every protein necessarily exhibits all possible structural features of the kinds just described. For instance, there are no disulfide bridges in myoglobin and hemoglobin, which are oxygen-storage and transport proteins and classic examples of protein structure, but they both contain Fe(II) ions as part of a prosthetic group. In contrast, the enzymes trypsin and chymotrypsin do not contain complexed metal ions, but they do have disulfide bridges. Hydrogen bonds, electrostatic attractions, and hydrophobic interactions occur in most proteins. It was pointed out earlier that the primary structure of a protein largely determines its higher level of structures. We can now see the reason for this relationship. When the particular side chains are in the proper positions, all of the hydrogen bonds, salt bridges, disulfide linkages, and hydrophobic interactions that stabilize the three-dimensional structure of that molecule can form. Altering the sequence of amino acid would mean that some of these forces might not be present, and thus, can cause destabilization of a protein structure. Consequently, when a protein is not in its proper structure, it cannot function normally. The proteins three-dimensional structure is determined by X-ray crystallography and, recently, by nuclear magnetic resonance (NMR) spectroscopy. Page 33 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department E. Quaternary Structure of a Protein The final level of protein structure is the quaternary structure and pertains only to multimeric proteins (protein composed of more than one polypeptide chain). The number of chains can range from 2 to more than a dozen, and the chains may be identical or different. Commonly occurring examples are dimers, trimers, and tetramers, consisting of two, three, and four polypeptide chains, respectively. The generic term for such a molecule, made up of a small number of subunits, is oligomer. The chains interact with one another non-covalently via electrostatic attractions, hydrogen bonds, and hydrophobic interactions. Allosteric Proteins As a result of non-covalent interactions in a proteins quaternary structure, subtle changes in structure at one site on a protein molecule may cause drastic changes in properties at a distant site. Proteins that exhibit this property are called allosteric. A classic illustration of the quaternary structure of proteins and its effect on properties is a comparison of hemoglobin, an allosteric protein, with myoglobin, which consists of a single polypeptide chain and is, therefore, not allosteric protein. Both hemoglobin and myoglobin bind to oxygen via a heme group. Hemoglobin Myoglobin Page 34 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department As shown in the illustration on the previous page, hemoglobin is a tetramer, a molecule consisting of four polypeptide chains: two α chains and two β chains. The two α chains of hemoglobin are identical, as are the two β chains. The overall structure of hemoglobin is α2β2 in Greek-letter notation. Both the α and β chains of hemoglobin are very similar to the myoglobin chain. The α chain is 141 residues long, and the β chain is 146 residues long. For comparison, the myoglobin chain is 153 residues long. Many of the amino acids of the α chain, the β chain, and myoglobin are homologous; that is, the same amino acid residues occupy the same positions. The heme group is the same in both myoglobin and hemoglobin. One molecule of myoglobin binds to one oxygen molecule. Four molecules of oxygen can bind to one hemoglobin molecule. Both hemoglobin and myoglobin bind oxygen reversibly, but the binding of oxygen to hemoglobin exhibits positive cooperativity, whereas oxygen binding to myoglobin does not. Positive cooperativity means that when one oxygen molecule is bound, it becomes easier for the next molecule to bind. A graph of the oxygen- binding properties of hemoglobin and myoglobin is one of the best ways to illustrate this point. When the degree of saturation of myoglobin with oxygen is plotted against oxygen pressure, a steady rise is observed until complete saturation is approached and the curve levels off. The oxygen- binding curve of myoglobin is thus said to be hyperbolic. In contrast, the oxygen-binding curve for hemoglobin is sigmoidal. This shape indicates that the binding of the first oxygen molecule facilitates the binding of the second oxygen, which facilitates the binding of the third oxygen, which in turn facilitates the binding of the fourth oxygen. This is precisely what is meant by the term “cooperative binding.” The two types of behavior are related to the functions of these proteins. Myoglobin has the function of oxygen storage in muscle. It must bind strongly to oxygen at very low pressures, and it is 50% saturated at a partial pressure of oxygen of 1 torr. The function of hemoglobin is oxygen transport, and it must be able both to bind strongly to oxygen and to release oxygen easily, depending upon conditions. In the alveoli of lungs (where hemoglobin must bind oxygen for transport to the tissues), the oxygen pressure is 100 torr. At this pressure, hemoglobin is 100% saturated with oxygen. In the capillaries running through active muscles, the pressure of oxygen is 20 torr, corresponding to less than 50% saturation of hemoglobin, which occurs at 26 torr. In other words, hemoglobin gives up oxygen easily in capillaries, where the need for oxygen is great. Page 35 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department V. PROTEIN HYDROLYSIS AND DENATURATION Hydrolysis of proteins causes disruption of the peptide bonds causing liberation of free amino acids. This can be carried out in the presence of strong acids, strong bases, and enzymes (proteases). Hydrolysis can be complete or partial. Denaturation, on the other hand, causes disruption (unfolding) of a protein’s three-dimensional structure due to the breakdown of the non-covalent interactions. Since the three-dimensional structure of proteins is very much related to their functions, denaturation, therefore, causes loss of biological activity. Denaturation can be reversible or irreversible. Irreversible denaturation results in coagulation and precipitation. The following are agents that can cause denaturation of proteins: Page 36 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department 1. Heat cleaves hydrogen bonds, so boiling a protein solution destroys the α-helical and β-pleated sheet structure. In collagen, the triple helixes disappear upon boiling, and the molecules have a largely random-coil conformation in the denatured state, which is gelatin. In other proteins, especially globular proteins, heat causes the unfolding of the polypeptide chains; because of subsequent intermolecular protein–protein interactions, coagulation then takes place. That is what happens when we boil an egg. 2. Addition of denaturing chemicals, such as 6 M aqueous urea, also breaks hydrogen bonds and causes the unfolding of globular proteins. 3. Surface active agents (detergents) change protein conformation by opening up the hydrophobic regions. 4. Acids, bases, and salts affect both salt bridges and hydrogen bonds. 5. Reducing agents, such as 2-mercaptoethanol can break disulfide bonds, reducing to - SH groups. The processes of permanent waving and straightening of curly hair are examples of the latter effect. The protein keratin, which makes up human hair, contains a high percentage of disulfide bonds. These bonds are primarily responsible for the shape of the hair, whether straight or curly. In either permanent waving or straightening, the hair is first treated with a reducing agent that cleaves some of the disulfide bonds. This treatment allows the molecules to lose their rigid orientations and become more flexible. The hair is then set into the desired shape, using curlers or rollers, and an oxidizing agent is applied. The oxidizing agent reverses the preceding reaction, forming new disulfide bonds, which now hold the molecules together in the desired positions. 6. Heavy metal ions (for example, Pb2+, Hg2+, and Cd2+) also denature protein by attacking the –SH groups. They form salt bridges, as in –S-Hg2+–S-. This very feature is taken advantage of in the antidote for heavy metal poisoning: raw egg whites and milk. The egg and milk proteins are denatured by the metal ions, forming insoluble precipitates in the stomach. These must be pumped out or removed by inducing vomiting. In this way, the poisonous metal ions are removed from the body. If the antidote is not pumped out of the stomach, the digestive enzymes would degrade the proteins and release the poisonous heavy metal ions, which would then be absorbed into the bloodstream. 7. Alcohol also denature proteins, coagulating them. This process is used in sterilizing the skin before injections. At a con-centration of 70%, ethanol penetrates bacteria and kills them by coagulating their proteins, whereas 95% alcohol denatures only surface proteins. Page 37 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department Unit 6 – CHEMISTRY OF ENZYMES INTENDED LEARNING OUTCOMES At the end of this unit, you will be able to: 1. Explain the role of for enzymes in biological systems; 2. Categorize enzymes based on reactions they catalyzed; 3. Explain different factors that can affect enzyme activity; 4. Explain the mechanisms as to how enzymes can catalyze biochemical reactions; and 5. Describe inhibition and regulation of enzymes. UNIT OUTLINE Topic Page I. General Characteristics of Enzymes A. Structural Features of Enzymes 38 B. Nomenclature of Enzymes C. Six Major Classification of Enzymes II. Enzymatic Activity A. How Enzymes Make Reaction Faster? B. Formation of the Enzyme-Substrate Complex 42 C. Nature of the Active Site D. Factors Affecting Enzyme Activity III. Enzyme Inhibition and Regulation A. Type of Enzyme Inhibition 46 B. Regulatory Mechanisms of Enzymes IV. Role of Enzymes in Medicine A. Enzyme Inhibitors Used as Drugs 51 B. Roles of Enzymes in Disease Diagnosis I. GENERAL CHARACTERISTICS OF ENZYMES One of the most important process in the human body is catalysis, which is the process of speeding up the rate of a chemical reaction by addition of a catalyst. Catalysts are not consumed during a reaction but merely help the reaction occur more rapidly. In the body, these catalysts are known as enzymes. Each cell in the human body comprises thousands of different enzymes because almost every reaction in a cell requires a specific enzyme. The term enzyme is derived from the Greek words en (meaning inside) and zyme (meaning yeast). Long before their chemical nature is fully understood, yeast enzymes were already used in the production of breads and alcoholic beverages. A. Structural Features of Enzymes 1. Enzymes are mostly globular proteins, except ribozymes which are RNA. Catalytic activities of enzymes depend on the integrity of their native conformation; thus, like proteins, their primary, secondary, tertiary, and quaternary structures are very Page 38 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department essential. If the enzyme is denatured or hydrolyzed, catalytic activity is lost. The factors that can cause denaturation of proteins also affect enzymes. 2. Enzymes are endowed with high catalytic power. Normal (non-enzymatic) catalysts increases the rate of a chemical reaction by a factor of 102 – 104, but enzymes, on the other hand, can increase the rate of biochemical reaction by a factor of 1020. Let us take the enzyme carbonic anhydrase as an example. This enzyme is responsible for the hydration of CO2 to form carbonic acid. As we know, this is an important reaction during respiration. The transfer of CO2 from the tissues to the blood and then to the air in the alveoli of the lungs would be less complete in the absence of the enzyme. In fact, carbonic anhydrase is one of the fastest enzymes known. Each enzyme molecule can hydrate 106 molecules of CO2 per second. carbonic anhydrase CO2 + H2O H2CO3 3. Enzymes are highly specific. Enzymes are highly specific both in the reactions the catalyze and in their choice of reactants, which are called substrates. An enzyme usually catalyzes a single chemical reaction or a set of closely related reactions. As an example, let consider the property of your proteases, which catalyzes proteolysis, the hydrolysis of a peptide bond. Papain, a protease found in papayas, hydrolyzes only peptide bonds but is quite undiscriminating of the identity of the adjacent side chains. This lack of specificity for side chain accounts for its use in meat-tenderizing sauces. On the other hand, trypsin which is a digestive enzyme we have encountered previously, is quite specific and catalyzes the splitting of peptide bonds only on the carboxyl side of lysine and arginine residues. Thrombin, an enzyme that participate in blood clotting, is even more specific than trypsin. It catalyzes the hydrolysis of Arg- Gly bonds in particular peptide sequences only. 4. Many enzymes require cofactors for activity. Some enzymes are composed of protein molecules only. They are considered as simple enzymes. The catalytic activity of many enzymes depends on the presence of small molecules termed cofactors, although the precise role varies with the cofactor and the enzyme. Generally, these cofactors are able to execute chemical reactions that cannot be performed by the standard set of twenty amino acids. An enzyme without its cofactor is referred to as an apoenzyme; the complete, catalytically active enzyme is called a holoenzyme. Holoenzyme Apoenzyme + Cofactor Page 39 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department Cofactors can be subdivided into two groups: (1) metals and (2) small organic molecules called coenzymes. Coenzymes are often derived from vitamins and can be either tightly or loosely bound to the enzyme. Tightly bound coenzymes are called prosthetic groups. Loosely associated coenzymes are more like cosubstrates because, like substrates and products, they bind to the enzyme and are released from it. The use of the same coenzyme by a variety of enzymes sets coenzymes apart from normal substrates, however, as does their source in vitamins. Enzymes that use the same coenzyme usually perform catalysis by similar mechanisms. B. Nomenclature of Enzymes Enzymes are commonly given names derived from the reaction that they catalyze and/or the compound or type of compound on which they act. For example, lactate dehydrogenase speeds up the removal of hydrogen from lactate (an oxidation reaction). Acid phosphatase catalyzes the hydrolysis of phosphate ester bonds under acidic conditions. As can be seen from these examples, the names of most enzymes end in “-ase.” Some enzymes, however, have older names, which were assigned before their actions were clearly understood. Among these are pepsin, trypsin, and chymotrypsin—all enzymes of the digestive tract. C. Six Major Classes of Enzymes Enzymes can be classified into six major groups according to the type of reaction they catalyze: 1. Oxidoreductases catalyze oxidations and reductions. Page 40 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department 2. Transferases catalyze the transfer of a group of atoms, such as from one molecule to another. 3. Hydrolases catalyze hydrolysis reactions. 4. Lyases catalyze the addition of two groups to a double bond or the removal of two groups from adjacent atoms to create a double bond. 5. Isomerases catalyze isomerization reactions. 6. Ligases, or synthetases, catalyze the joining of two molecules with a concomitant usage of ATP as energy source. Page 41 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department II. ENZYMATIC ACTIVITY A. How Do Enzymes Make Reactions Faster? A chemical reaction of substrate S to from product P goes through a transition state X‡ that has higher free energy than does either S or P. S X‡ P The transition state is a transitory molecular structure that is no longer the substrate but is not yet the product. The transition state is the least stable species in a reaction pathway due to its high free energy. The difference in free energy between the transition state and the substrate is called the activation energy, denoted as ΔG‡. Enzymes, like any catalysts, make the reaction faster by facilitating the formation of the transition state, or in other words, lowering the activation energy. The combination of substrate and enzyme creates a reaction pathway whose transition state energy is lower than that of the reaction in the absence of enzyme. Because the activation energy is lower, more molecules have the energy required to reach the transition state. B. Formation of the Enzyme-Substrate Complex The first step in catalysis is the formation of an enzyme-substrate (ES) complex. Substrates bind to a specific region of the enzyme called the active site. Most enzymes are highly selective in the substrates that they bind. In an enzyme catalyzed reaction, the enzyme E binds to the substrate S to form the enzyme-substrate (ES) complex. The formation of the complex leads to the formation of the transition state, which then forms the product P. E+S ES E+P What evidence support the theory of ES formation? One evidence of the formation of the ES complex was the observation that, at constant concentration of enzyme, the reaction rate increases with increasing substrate concentration until a maximal velocity is reached. The graph on the left having reaction velocity as y-axis and substrate concentration as x-axis demonstrate this observation. In contrast, non- enzymatic reactions do not show this saturation effect. The fact that an enzyme-catalyzed reaction has a maximal velocity suggests the formation of a discrete ES complex. Page 42 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department At sufficiently high substrate concentration, all the catalytic sites are filled or saturated, and so the reaction rate cannot increase. Models of Binding Substrate to Enzyme Substrate can bind to the enzyme because of highly specific interactions between the substrate and the side chains and backbone groups of amino acids making up the active sites. Two important models have been developed to describe the binding process – the lock- and-key model and induced fit model. 1. The lock-and-key model is developed by Emil Fischer which assumes high degree of similarity between the shape of the substrate and the geometry of the active site on the enzyme. The substrate binds to a site whose shape complements its own, like a key in a lock or the correct piece in a three-dimensional jigsaw puzzle. This model has intuitive appeal but is now largely of historical interest because it does not take into account an important property of proteins, namely their conformational flexibility. 2. The induced fit model takes into account the fact that proteins have some three- dimensional flexibility. According to this model, the binding of the substrate induces a conformational change in the enzyme that results in a complementary fit after the substrate is bound. The binding site has a different three-dimensional shape before the substrate is bound. C. Nature of the Active Site The active site of an enzyme is the region that binds the substrate (and the cofactor, if any). It also contains the amino acid residues that directly participate in the making and breaking of bonds. These residues are called the catalytic groups. In essence, the interaction of the enzyme and substrate at the active site promotes the formation of the transition state. Page 43 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department The active site is the region of the enzyme that most directly lowers activation energy of the reaction, thus providing the rate-enhancement characteristic of enzyme action. Recall that proteins are not rigid structures, but are flexible and exist in an array of conformations. Thus, the interaction of the enzyme and substrate at the active site and the formation of the transition state is a dynamic process. Although enzymes differ widely in structure, specificity, and mode of catalysis, a number of generalizations concerning their active sites can be stated: 1. The active site is a three-dimensional cleft, or crevice, formed by groups that come from different parts of the amino acid sequence: indeed, residues far apart in the amino acid sequence may interact more strongly than adjacent residues in the sequence, which may be sterically constrained from interacting with one another. For example, in lysozyme, an enzyme that degrades the cell walls of some bacteria, the important groups in the active site are contributed by residues numbered 35, 52, 62, 63, 101, and 108 in the sequence of 129 amino acids (See figure below). 2. The active site takes up a small part of the total volume of an enzyme. Although most of the amino acid residues in an enzyme are not in contact with the substrate, the cooperative motions of the entire enzyme help to correctly position the catalytic residues at the active site. Experimental attempts to reduce the size of a catalytically active enzyme show that the minimum size requires about 100 amino acid residues. In fact, nearly all enzymes are made up of more than 100 amino acid residues, suggesting that all amino acids in the protein, not just those at the active site, are ultimately required to form a functional enzyme. 3. Active sites are unique microenvironments. In all enzymes of known structure, active sites are shaped like a cleft, or crevice, to which the substrates bind. Water is usually excluded unless it is a reactant. The nonpolar microenvironment of the cleft enhances the binding of substrates as well as catalysis. Nevertheless, the cleft may also contain polar residues, some of which may acquire special properties essential for substrate binding or catalysis. The internal positions of these polar residues are biologically crucial exceptions to the general rule that polar residues are located on the surface of proteins, exposed to water. Page 44 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department D. Factors Affecting Rate of Enzymatic Reaction 1. Concentration of Enzymes The reaction rate of an enzymatic reaction is directly proportional to the concentration of the enzyme. 2. Temperature - As the temperature of an enzymatic reaction increases, so is the rate of the reaction. However, when the temperature increases beyond a certain point, the increased energy begins to denature the enzyme which impedes catalytic action. The enzyme activity quickly decreases as the temperature climbs past this point. The temperature at which enzyme has maximum activity is known as the enzyme’s optimum temperature. 3. pH - The pH of enzyme’s environment affects its activity because the charge on acidic and basic amino acids located on the active site is dependent on pH. Small changes in pH (less than 1 unit) can result in enzyme denaturation and subsequent loss of activity. For this reason, most enzymes exhibit maximum activity over a narrow pH range. The temperature at which enzyme has maximum activity is known as the enzyme’s optimum temperature. Page 45 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department III. ENZYME INHIBITION AND REGULATION A. Types of Enzyme Inhibition An inhibitor is any substance that slows or stops the normal catalytic function of enzymes by binding to it. Inhibition of enzymes may be reversible or irreversible. There 3 types of reversible inhibition – competitive, non-competitive, and uncompetitive. Irreversible Inhibition During irreversible inhibition of an enzyme, the inhibitor forms a strong covalent bond to an amino acid side chain at the enzyme’s active site which inactivates the enzyme. Addition of excess substrate does not reverse this inhibition process as the formed inhibitor-active site bond is sufficiently strong. Thus, the enzyme is permanently deactivated. Organophosphate insecticides are based on irreversible inhibition. Reversible Inhibition Unlike in irreversible inhibitors, reversible inhibitors do not form strong covalent bonds with the active site. They can be dissociated when certain factors are adjusted. There are 3 types of reversible inhibition: 1. Competitive Inhibition A competitive inhibitor is a molecule that sufficiently resembles an enzyme substrate in shape and charge distribution that it can compete with the substrate for occupancy of the enzyme’s active site. When a competitive inhibitor binds to an enzyme active site, an enzyme-inhibitor complex is formed. The inhibitor remains unchanged when bound to the active site, but its physical presence at the site prevents the normal substrate molecule from occupying the site. This results to decrease in enzyme activity. The formation of enzyme-inhibitor complex is reversible process since it is only maintained by weak interactions. With time (a fraction of a second), the complex breaks up. The empty active site is then available for another occupant. Substrate and inhibitor again compete for the empty active site. Thus, the active site of an enzyme binds either inhibitor or normal substrate on a random basis. Competitive inhibition can be reduced by simply increasing the substrate concentration. Treatment of methanol poisoning by giving intravenous ethanol is based on the principle of competitive inhibition. Page 46 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department 2. Non-competitive Inhibition A non-competitive inhibitor is a molecule that binds to a site on an enzyme other than the active site. The presence of this inhibitor causes a change in the conformation of an enzyme which prevents the catalytic groups at the active site from properly effecting their catalytic action. Increasing substrate concentration does not completely overcome the inhibitory effect of a non-competitive inhibitor. Lowering the concentration of the inhibitor sufficiently does free up many enzymes, which then return to normal activity. One example of non-competitive inhibition is given by glucose-6-phosphate inhibiting hexokinase in the brain. Carbons 2 and 4 on glucose-6-phosphate contain hydroxyl groups that attach along with the phosphate at carbon 6 to the enzyme-inhibitor complex. The substrate and enzyme are different in their group combinations that an inhibitor attaches to. The ability of glucose-6-phosphate to bind at different places at the same time makes it a non-competitive inhibitor. 3. Uncompetitive Inhibition Uncompetitive inhibition takes place when an enzyme inhibitor binds only to the enzyme-substrate complex, not on the free enzyme. The binding site of an uncompetitive inhibitor is created only on interaction When uncompetitive inhibitor binds to the ES complex, it stabilizes the complex making it more difficult for substrate to dissociate or be converted to product. B. Regulatory Mechanism of Enzymes Regulation of enzyme activity within a cell is very necessary for many reasons. For example: 1. A cell that continually produces large amount of enzymes for which substrate concentration is always very low is wasting energy. The production of the enzyme needs to be “turned off”. 2. A product of an enzyme-catalyzed reaction that is present in plentiful amounts in cell is a waste of energy if the enzyme continues to catalyze the reaction that produces the product. The enzyme needs to be “turned off.” Page 47 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department Allosteric Enzymes Many, but not all, enzymes responsible for regulating cellular process are allosteric enzymes. The following are characteristics of these enzymes: 1. Have quaternary structures 2. Have 2 binding sites, for substrate and for regulators. These 2 binding sites are distinct from each other in terms of location and shape. Substances that bind to the regulatory site are called regulators. Regulators can be positive regulators (increases enzyme activity) or negative regulators (non-competitive inhibitors). Different Mechanisms of Enzyme Control 1. Feedback Control - Most biochemical processes within cells take place in several steps rather than a single step. A different enzyme is required for each step of the process. Feedback control is a process in which activation or inhibition of the first reaction in a reaction sequence is controlled by a product of a reaction sequence. 2. Proteolytic Activation of Proenzymes Proenzymes (zymogens) are inactive form of an enzyme. The inactive form of enzymes can be turned on at the appropriate time by cleavage of one or a few specific peptide bonds. This activation is irreversible and occurs once in the life of an enzyme molecule. Specific proteolysis is the common means of activating these proenzymes. For example: a. Digestive enzymes that are synthesize in the stomach and pancreas are synthesized as proenzymes. Page 48 of 142  CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department The digestion of proteins and other molecules in the duodenum requires the concurrent action of several enzymes, because each is specific for a limited number of side chains. Thus, the zymogens must be switched on at the same time. Coordinated control is achieved by the action of trypsin as the common activator of all the pancreatic zymogens — trypsinogen, chymotrypsinogen, proelastase, procarboxypeptidase, and prolipase, the inactive precursor of a lipid-degrading enzyme. To produce active trypsin, the cells that line the duodenum display a membrane-embedded enzyme, enteropeptidase, which hydrolyzes a unique lysine–isoleucine peptide bond in trypsinogen as the zymogen enters the duodenum from the pancreas. The small amount of trypsin produced in this way activates more trypsinogen and the other zymogen. Thus, the formation of trypsin by enteropeptidase is the master activation step. b. Blood clotting is mediated by cascade of proteolytic activation that ensures a rapid and amplified response to trauma. Shown above is the diagram for the blood-clotting cascade. Inactive forms of clotting factors are shown in red; their activated counterparts are in yellow. Stimulatory proteins that are not themselves enzymes are shown in blue boxes. Fibrin clot is formed by the interplay of the intrinsic, extrinsic, and final common pathways. The intrinsic pathway begins with the activation of factor XII (Hageman factor) by