Module 4 Chemistry of Proteins PDF

Republic of the Philippines ISABELA STATE UNIVERSITY Echague, Isabela COLLEGE OF ARTS AND SCIENCES First Semester, School Year 2024-202...

Republic of the Philippines ISABELA STATE UNIVERSITY Echague, Isabela COLLEGE OF ARTS AND SCIENCES First Semester, School Year 2024-2025 Biochemistry Module 4 I. Chemistry of Proteins INTRODUCTION Proteins are the most abundant organic molecules in cells, constituting 50 percent or more of their dry weight. They are very varied and complex. Millions of different proteins exist in the biosphere as each species of life possesses a chemically distinct group of proteins The presence of proteins in almost every part of the living organism eloquently shows the variety of functions they play. Proteins serve as the catalysts for biological reactions and as structural elements that dictate much of as antibodies, they are the defense system of the body, and as hormones they regulate the body's glandular activity. In the blood they maintain fluid balance, are part of the clotting process, and transport oxygen and lipids. They can act as poisons, like the venoms in animal bites, or toxins, like the bacterial toxin causing botulism in improperly produced and some antibiotics that are secretions of bacteria are protein in nature. A single cell can contain thousands of proteins, each with a unique function. Although their structures, like their functions, vary greatly, all proteins are made up of one or more chains of amino acids. In this chapter, we will look in more detail at the building blocks, structures, and roles of proteins. LEARNING OUTCOMES At the end of this unit, you should be able to: 1. describe the general structure of amino acids 2. classify the twenty amino acids according to their differences in structure, charges and polarity 3. illustrate the amphoteric nature of amino acids 4. illustrate how amino acids polymerize into proteins 5. classify proteins according to structure 6. discuss the four levels of protein organization LEARNING CONTENTS 1. AMINO ACIDS: BUILDING BLOCKS OF PROTEINS Amino acids are the monomers that make up proteins. Specifically, a protein is made up of one or more linear chains of amino acids, each of which is called a polypeptide. There are 20 types of amino acids commonly found in proteins. Any organic molecule with at least one CARBOXYL group (organic acid) and at least one AMINO group (organic base) Type formula is represented below: Figure 4.1 Type formula of amino acid Amino acids share a basic structure, which consists of a central carbon atom, also known as the alpha (α) carbon, bonded to an amino group -NH2, a carboxyl group -COOH, and a hydrogen atom. Although the generalized amino acid shown above is shown with its amino and carboxyl groups neutral for simplicity, this is not actually the state in which an amino acid would typically be found. At physiological pH (7.27 - 7.47), the amino group is typically protonated and bears a positive charge, while the carboxyl group is typically deprotonated and bears a negative charge. Every amino acid also has another atom or group of atoms bonded to the central atom, known as the R group, which determines the identity of the amino acid. For instance, if the R group is a hydrogen atom, then the amino acid is glycine, while if it’s a methyl (-CH3), the amino acid is alanine. The twenty common amino acids are shown in the figure below.  Figure 4.2. Structures of the 20 amino acids AMINO ACID ABBREVIATIONS This table shows the abbreviations and single letter codes used for the 20 amino acids found in proteins.. Table 4.1. List of amino acids and their codes  ESSENTIAL AMINO ACIDS The human body is able to synthesize 11 of the 20 amino acids, however the other nine we cannot. This is likely as a result of gene loss or mutation over time in response to changing selective pressures, such as the abundance of particular food containing specific amino acids. These are therefore termed essential amino acids and must be acquired through our diet. Particular animal species are able to synthesize different amino acids and, accordingly, their dietary requirements differ. Humans for example are able to synthesize arginine, but dogs and cats cannot – they must acquire it through dietary intake. Unlike humans and dogs, cats are unable to synthesize taurine. This is one of the reasons that commercial dog food is unsuitable for cats. For humans, the nine amino acids that must be acquired through diet are histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Foods that contain all nine essential amino acids are referred to as "complete proteins", and include meat, seafood, eggs, dairy products, soy, quinoa and buckwheat. Other protein sources, such as nuts, seeds, grains and beans, contain some but not all essential amino acids and are therefore referred to as incomplete. Table 4.2 Naturally occurring amino acids 2. THE L-FAMILY Amino acids can occur in L- and D-forms, but only L-forms are used by cells. Every amino acid (except glycine) can occur in two isomeric forms, because of the possibility of forming two different enantiomers (stereoisomers) around the central carbon atom. By convention, these are called L- and D- forms, analogous to left-handed and right-handed configurations (Figure 4.3). Figure 4. 3. The D & L amino acids Only L-amino acids are manufactured in cells and incorporated into proteins. Some D- amino acids are found in the cell walls of bacteria, but not in bacterial proteins. Glycine, the simplest amino acid, has no enantiomers because it has two hydrogen atoms attached to the central carbon atom. Only when all four attachments are different can enantiomers occur. 1. ACID-BASE PROPERTIES OF THE AMINO ACIDS The α-COOH and α-NH2 groups in amino acids are capable of ionizing (as are the acidic and basic R-groups of the amino acids). As a result of their ionizability the following ionic equilibrium reactions may be written: R-COOH ↔ R-COO– + H+ R-NH3+ ↔ R-NH2 + H+ The equilibrium reactions, as written, demonstrate that amino acids contain at least two weakly acidic groups. However, the carboxyl group is a far stronger acid than the amino group. At physiological pH (around 7.4) the carboxyl group will be unprotonated and the amino group will be protonated. An amino acid with no ionizable R-group would be electrically neutral at this pH. This species is termed a zwitterion.  Like typical organic acids, the acidic strength of the carboxyl, amino and ionizable R- groups in amino acids can be defined by the association constant, Ka or more commonly the negative logrithm of Ka, the pKa. The net charge (the algebraic sum of all the charged groups present) of any amino acid, peptide or protein, will depend upon the pH of the surrounding aqueous environment. As the pH of a solution of an amino acid or protein changes so too does the net charge. This phenomenon can be observed during the titration of any amino acid or protein. When the net charge of an amino acid or protein is zero the pH will be equivalent to the isoelectric point: pI. 2. ISOELECTRIC POINT The isoelectric point of any amino acid is the pH value at which the amino acid exists predominantly in its neutral zwitterion form. At the isoeletric point, the concentration of the ammonium ion form (positive charge) is equal to the concentration of the carboxylate ion form (negative charge). This implies that the net charge on the amino acid at the isolectric point is zero. The side chain group, usually designated with the letter R, determines the isoelectric point. Since most R-groups differ from one amino acid to the other, the isoelectric points will also differ. The isoelectric points (IP) of amino acids range from 2.8 to 10.8, Glycine, with an IP of 6.0 exist as a positively charged species at a pH below 6.0. For instance, glycine has an isoelectric point of about 6.0 while arginine has an isoelectric point of about 10.8. This difference is a result of the difference in the side groups - arginine has a much more acidic side group and this will bump the isoelectric point up. In solutions that are more basic than 6.0, solutions with a pH>6.0 the NH3+ loses a proton and the amino acid has a negative charge. Glycine, with a IP of 6.0, has a 1- charge in solutions that have a pH above 6.0.  At a given fixed pH, any amino acid will have its own unique concentration of positively and negatively charged forms. When the mixture of amino acids are placed onto a fluid or paper and a constant electric field is applied, the negatively charged amino acids will move to the anode (positive side) while the positively charged amino acids will move to the cathode (negative side). On this basis the proteins are separated from each other at different pH values by a process called electrophoresis. Electrophoresis is a process that separates a mixture of amino acids by the nature of their isoelectric point. This technique has been a useful tool in the analysis of proteins in blood serum. At the isoelectric point (IP), the amino acids and protein exhibit minimum solubility and will start to solidify out of the solution. For casein, the IP is approximately 4.6 and it is the pH value at which acid casein is precipitated. In milk, which has a pH of about 6.6, the casein micelles have a net negative charge and are quite stable. During the addition of acid to milk, the negative charges on the outer surface of the micelle are neutralized (the phosphate groups are protonated), and the neutral protein precipitates. The same principle applies when milk is fermented to curd. The lactic acid bacillus produces lactic acid as the major metabolic end-product of carbohydrate [lactose in milk] fermentation. The lactic acid production lowers the the pH of milk to the IP of casein. At this pH, casein precipitates. 3. BUFFERING PROPERTIES Because amino acids can act both as acids and bases, they are effective buffers in aqueous solutions. In the body amino acids and proteins help maintain blood pH within its very narrow normal range. Having a working knowledge of amino acids, let us look at the very special polymers they form, the proteins. A naturally occoring protein that is biologically active has a highly complex three dimensional structure. There are four levels of structure that interdependently influence a proteins native conformation. They are called primary, secondary, tertiary and quarternary structures. 4. LEVELS OF STRUCTURE Primary Structure This level of structure refers to the number and sequence of amino acids in a protein. The feature responsible for the primary structure is the covalent peptide bond that joins amino acids in a protein. The peptide bond is an amide bond formed by joining the carboxyl group of one amino acid to the amino group of another amino acid simultaneous to the removal of water is shown. Figure 4-4. Formation of a peptide bond The peptide bond is a stable bond it can be broken cnly by ocid or bese hydrolysis or by specific enzymes. The compounds produced by linking amino acids are known as peptides; two amino acids form a dipeptide, three forms tripeptide, end more then three, a polypeptide Long polypeptide are called proteins There are standard rules for naming peptides and proteins. Because proteins contain very large numbers amino acids, the three-letter abbreviations of the amino acid names given are used when writing the amino acid sequence. The peptide bond is represented by dash between the amino acid names. The amino acid that contains the free amino group is called N- terminal amino acid and is written first, while amino acid with the free carboxyl group, called C-Terminal amino acid is written last. The following is an example of these naming rules: Valine - aspartic acid – alanine- tryptophan-leucine val - asp - ala – trp - leu (N-terminal) (C-terminus) Another example :  It can be safely stated that the primary structure determines the biological role of a protein and is critical for its proper functioning There ore observations that changing just one amino acid in a sequence can disrupt the entire protein molecule For example, hemoglobin, the red colored protein in blood responsible for oxygen transport has a total of 574 amino acids. Changing just one of these specifically glutamic acid at position 6 by valine results in the defective hemoglobin molecule found in patients with sickle cell anemia The determination of the amino acid sequence of a protein is a complex procedure that was first developed by F. Sanger in 1953. But before any attempt at sequencing can be done, the amino acid composition of a given protein must first be determined. This can be done by either hydrolyzing the protein or allowing it to be digested by proteolytic enzymes, and then determining the type and amount of amino acids present by ion exchange chromatography, a procedure that capitalizes on the different ionization characteristics of the individual amino acids. Once the amino acid composition has been established determining the amino acid sequence can now be done. The original protein is broken down by specific chemicals or enzymes into smaller peptide units from which the sequence is then deduced. The process of sequencing is best explained by the following examples. A peptide yields the following smaller peptides upon digestion with the enzymes trypsin and chymotrypsin. a. trypsin - catalyzes the hydrolysis of peptide bonds involving the carboxyl groups of basic amino acids. peptide 1: ala- lys peptide 2: ala-phe-ser-gly, peptide 3: asp-tyr-arg b. chymotrypsin - catalyzes the hydrolysis of peptide bonds involving the carboxyl group of aromatic amino acids peptide 1: ala-lys-asp-tyr peptide 2 phe-ser- gly peptide 3: arg-ala-phe  To establish the sequence of amino acids in the original peptide, the simplest way is to fit each small peptide to form a continuous sequence, like fitting pieces in a Jigsaw puzzle. ala-lys ala-lys-asp-tyr asp-tyr-arg arg-ala-phe ala-phe-ser-gly phe-ser-gly The sequence of amino acids therefore in the original peptide is ala-lys-asp-tyr-arg-ala-phe-ser-gly. Secondary Structure The secondary structure of a protein refers toa reguler geometric pattern along a polypeptide brought about by H-bonding involving peptide bonds. Two arrangements were established by Linus Pauling by means of X-ray diffraction They are the ὰ-helix and the pleated sheet.  Figure 4.5 The ὰ-helix In this arrangement the amino acids are coiled resembling a circular staircase with the loops held together by H-bonds represented as dotted lines (Figure 4.4 ). There are 36 amino acids per turn of the alpha helix and the R-groups extends outward from the helix. Hair and wool are made up of several coils of keratin wound around each other like ropes and held together by disulfide bridges (Fig.4.6 ). Figure 4.6 The coils of keratin Pleated sheet or β-conformation  A beta-pleated sheet (β-pleated sheet) is a secondary structure that consists of polypeptide chains arranged side by side; it has hydrogen bonds between chains has R groups above and below the sheet is typical of fibrous proteins such as silk. Figure 4.7 The beta pleated sheet Tertiary Structure: Globular Proteins Figure 4.8 Tertiary structure of proteins This level of structure refers to the folding and coiling of a polypeptide to produce a complex, globular molecule shape. At physiological pH and temperature this shape is most thermodynamically stable and is referred to as the native state of protein. The folding and coiling a -protein undergoes is specified by tho particular set and sequence of amino acids and is a result of group interactions, the most prominent of which are the following:  1. H-bonds between the atoms in the R groups of amino acids 2. Salt bridges or ionic interactions between ionized acidic and basic amino acids 3. Hydrophobic interactions between non polar groups 4. Disulfide linkages between two cysteine amino acids 5. Hydrophilic attractions between polar or ionized group and water on the surface of the tertiary structure Quaternary structure Proteins that contain more than one polypeptide chain ore known as oligomeric proteins. The manner in which the separate polypeptide chains fit together to form biologically active unit is referred to.as the quaternary „structure. Hydrogen bonding, hydrophobic interactions and salt bridges may be involved in holding the coins in position. Hemoglobin is one example of a protein with o quaternary structure as shown in Figure 4.9. Figure 4.9 Quaternary structure of hemoglobin It consists of four polypeptide chains, two alpha chains and two beta chains arranged around an Iron.containing heme group. Obviously, a protein consisting of only one polypeptide chain does not have a quaternary structure. 7. DENATURATION Proteins are defined by their primary, secondary, tertiary and for some, quaternary structures. They give the proteins cartoon identifying properties like biological enzymatic, solubility, ionic, reactivity of R-group molecular weight and size. Disruption of this native structure of the protein is called denaturation. This process involves the uncoiling of the protein molecule which causes the loss of its biological! activity. This is shown in Fig. 4.9 primary structure intact. SUMMARY Proteins are composed of amino acids bound together by peptide linkages. An amino acid is a molecule having both a carboxyl group and an amino group making it an amphoteric substance. It also has a chemically distinct side chain or R-group. There are 20 different R- groups and therefore 20 different amino acids that are required by organisms for protein synthesis.  There are four levels of protein structure - primary secondary, tertiary and quaternary. Primary structure is the definite amino acid sequence along a polypeptide; secondary structure refers to other the helix or pleated sheet conformation made possible by maximum H-bonding involving peptide linkages; tertiary structure is to the three-dimensional folding and bending of a polypeptide into a compact globular shape due to R-group interactions, and quaternary structure of oligomeric proteins which describes the arrangement of 2 or more polypeptides as a single biologically active unit. Various changes in the surroundings of a protein can disrupt the complex secondary, tertiary and quaternary structure of the molecule. Disruption of the native state of the protein is called denaturation. It can cause the loss of any ot the properties ascribed to its native state including its biological activity. Among the more common denaturants are heat, extremes in pH, heavy metals, organic solvents and reducing agents. KEY TERMS/CONCEPTS amino acids electrophoresis hydrophobic casein essential number sequence nonessential peptide bond amphoteric ion exchange chromatography zwitterion helix isoelectric point pleated sheet FLEXIBLE TEACHING LEARNING MODALITY (FTLM) Module, Exercises and Google Meet TEACHING AND LEARNING ACTIVTIES (See Biomolecules Laboratory Manual) LEARNING MATERIALS AND RESOURCES Video ▪ Protein Function and Malfunction in Cells : https://www.ncbi.nlm.nih.gov/books/NBK21297/ ASSESSMENT TASK I. True/False 1. Amino acids found in nature are generally the L-forms 2. At the isoelectric pH of an amino acid, its solubility in water is maximum. 3. A basic amino acid such as asparagine would have a net negative charge at high pH. 4. The amino acid glycine is the C-terminal amino acid of the pentapeptide Gln-Asp-Pro-Val- Gly. 5. In aqueous solutions, the hydrocarbon side chains of a protein tend to point inward, avoiding interaction with the water. 6. Protein denaturation is always irreversible. 7. The amino acid proline disrupts the alpha-helical secondary structure of a protein. 8. Myoglobin is used for oxygen storage in the muscle. 9. Proteins are composed of amino acids. 10. All protein molecules have a quaternary structure. II. Match the following. Each answer will be used once only a. Fibrous protein 1. water-soluble protein, easily denatured b. Native state 2. can be reversible or irreversible c. disulfide bridge 3. the metal ion of a conjugated protein d. peptide bond 4. a protein in its normal, biologically active form e. globular protein 5. ionic bond between carboxylate ion and protonated amine group f. denaturation 6. important structural protein g. prosthetic group 7. covalent bond that can hold two peptide chains together h. salt bridge 8. protein that has a prosthetic group i. conjugated 9. covalent bond that holds two amino acids together protein REFERENCES Berg, J.M., Tymoczko, J.L. and Stryer, L. (2011) Biochemistry. Freemann, 7th edition, retrieved at http://www.whfreeman.com Brown, P. (2009). Quick Reference to Wound Care, James and Bartelt. Publishing Chulay, M. (2011).ACCN Essentials of Critical Care Nursing. McGraw-Hill Publishing Hein, Morris (2005). Introduction to General, Organic and Biochemistry, John Wiley and Sons Jones, Evangeline. (2011). Manual of Practical Medical Biochemistry. Moore, John (2008). Chemistry: The Molecular Science. Thomson Brooks Nelson, D.L. & Cox, M.M. Lehninger Principles of Biochemistry. Freeman, 6th edition http://bcs.whfreeman..com./lehninger6e/#t_824263/ Silberberg, M. (2010). Priciples of General Chemistry. Mc Graw Hill Voet, J. and Judith Voet. (2011) Biochemistry, 4th Edition. 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A three-pronged program, targeting education, close follow-up and a dietary supplement, significantly decrease body weight and body fat - DOI: 10.15761/IOD.1000143 The Birth Control Pill.- https://embryo.asu.edu/pages/birth-control-pill Genetic medicines: treatment strategies for hereditary disorders - Timothy P. O'Connor & Ronald G. Crystal Nature Reviews Genetics volume 7, pages261–276 (2006 Energy and Life - http://www.BioLerner.com Energy and Life: The Transformation of Energy in Living Organisms - https://study.com/academy/lesson/energy-and-life-the-transformation-of-energy-in-living- organisms.html ISUE_-CAS-DSM-016 Revision: 1 Effectivity: August 1, 2020