Amino Acids PDF

AMINO ACIDS AND PRIMARY STRUCTURE OF PROTEINS FUNCTIONS OF PROTEINS 1. catalysts - enzymes for metabolic pathways 2. storage and transport - e.g. myoglobin and hemoglobin 3. structural - e.g. actin, myosin 4. mechanical work - movement of flagella and cilia, microtubule...

AMINO ACIDS AND PRIMARY STRUCTURE OF PROTEINS FUNCTIONS OF PROTEINS 1. catalysts - enzymes for metabolic pathways 2. storage and transport - e.g. myoglobin and hemoglobin 3. structural - e.g. actin, myosin 4. mechanical work - movement of flagella and cilia, microtubule movement during mitosis, muscle contraction 5. decoding information - translation and gene expression 6. hormones and hormone receptors 7. specialized functions - e.g. antibodies STRUCTURE OF AMINO ACIDS There are 20 common amino acids called α-amino acids because they all have an amino (NH3+) group and a carboxyl group (COOH) attached to C-2 carbon (α carbon).H α H C COO carbon NH3 At pH of 7, amino group is protonated (-NH3+) and carboxyl group is ionized (COO-). The amino acid is called a zwitterion. pKa of a carboxyl group = 1.8 - 2.5 pKa of a amino group = 8.7 - 10.7 H H H C COOH H C COO NH2 NH3 zwitter ion The α carbon is chiral or asymmetric ( 4 different groups are attached to the carbon; exception is glycine.) chiral H H H C COO H3C C COO NH3 NH3 GLYCINE L- ALANINE Amino acids exist as STEREOISOMERS (same molecular formula, but differ in arrangement of groups). DIASTEREOMER Amino acids used in nature are of L configuration. S carboxylate group at top --> points away side chain at bottom α amino group orientation determines NH3+ on left = L NH3+ on right = D Designated D(right) or L(left). COOH COOH H C NH2 H2N C H CH3 CH3 DEXTRO- LEVO- configuration configuration If -NH2 is below α-carbon and –COOH is at the right of it STRUCTURES OF 20 COMMON AMINO ACIDS: Amino acids are grouped based upon the properties and structures of side chains. 1. Amino acids with an aliphatic side chain O O a. glycine b. L- alanine H2N CH C OH H2N CH C OH CH3 H c. L- valine d. L- isoleucine e. L-leucine O O O H2N CH C OH H2N CH C OH H2N CH C OH CH CH3 CH2 CH CH3 CH2 CH CH3 CH3 CH3 CH3 2. Amino acids with aromatic branch a. L-tryptophan b. L-tyrosine O H2N CH C OH O CH2 H2N CH C OH CH2 c. L-phenylalanine HN OH O H2N CH C OH CH2 3. Amino acids with polar –OH group a. serine b. L- threonine O O H2N CH C OH H2N CH C OH CH2 CH OH OH CH3 4. Amino acids containing sulfur a. L- cysteine b. L- methionine O O H2N CH C OH H2N CH C OH CH2 CH2 CH2 SH S CH3 5. Amino acids with amide group a. L- glutamine b. L-asparagin O O H2N CH C OH H2N CH C OH CH2 CH2 CH2 C O C O NH2 NH2 6. Imino acids a. L-proline b. L-hydroxyproline O O C OH C OH HN HN OH 7. Amino acids that are basic a. L-Arginine b. L-lysine c. L-hydroxylysine O O O H2N CH C OH H2N CH C OH H2N CH C OH CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 HC OH NH CH2 CH2 C NH NH2 NH2 NH2 8. Acidic amino acids a. L-histidine b. L-glutamic acid c. L-aspartic acid O O O H2N CH C OH H2N CH C OH H2N CH C OH CH2 CH2 CH2 CH2 N C O C O NH OH OH PROPERTIES 1. Amino acids are generally soluble in water, insoluble in non-polar solvents like benzene and ether. 2. AA have high MP (above 200ᵒC) and low vapor pressure. 3. AA have large dipole moments and high dielectric constants. O + H3N CH C O + -- -- H 4. Except for glycine, the α carbon is chiral and therefore capable of optical isomerism. 5. Aromatic amino acids absorb light in the ultraviolet region, and this property is useful for estimating protein content. e.g. phe absorbs 260nm, trp at 280,tyr at 275nm CHEMICAL PROPERTIES 1. acid-base properties. Amino acids are amphiprotic. a. the –COOH group b. the protonated amine grp NH3 c. –SH grp of cysteine d. protonated nitrogen of the heterocyclic ring of histidine e. phenolic –OH of tyrosine  2. Zwitterionic character of amino acids O O O pk2=9.7 pk1= 2.3 H3N CH C OH H3N CH C O H2N CH C O pI  CH3 CH3 CH3 Ala+ Alaᵒ Ala¯ pI = pk1 + pk2 2 pI = 6.0 3. Reactions of the –COOH group a. Esterification (RCOOR) O O H2N CH C OH C2H5OH H2N CH C OC2H5 H2O H H b. Acylation O O PCl3 H3N CH C O H3N CH C Cl POCl3 H H 4. Decarboxylation rxns- O H2N CH C OH H2N CH2 CH2 CH2 N N NH NH COLOUR REACTIONS OF AMINO ACIDS AND PROTEINS 1. Biuret test This is the test indicates the presence of peptide linkages. The purplish to violet colour is apparently due to the cupric ions with the unshared electron pairs of four nitrogen atoms. All substance is proportional to the number of peptide bonds give the test and the intensity of the purple colour produced id proportional to the number of peptide bonds present.  Ninhydrin test  Amino acids reacts with ninhydrin (tryketoninhydrindene hydrate) to yield CO 2,NH3 and aldehyde containing one less carbon than the amino acid. The reaction is also yields a blue or purple colour useful for the colorimetric determination of amino acids.  Xanthoproteic test  This test is positive for proteins and amino acids containing an aromatic side chain(phenylalanine, tyrosine, and tryptophan). The benzene ring undergoes nitration with concentrated HNO3giving nitro derivatives which are yellow in colour. Phenylalanine does not response readily to this test and requires H2SO4 as catalyst.  Millon-Nasse test  The phenolic group of tyrosine reacts with Millon-Nasse reagent (HgSO4 in H2SO4)forming an old rose or pink to red complex upon heating. The complex is probably the mercury salt of the phenolic compound. SEPARATION AND CHARACTERIZATION TECHNIQUES FOR PROTEINS AND AMINO ACIDS  1. FUNCTION Proteins perform a large variety of functions:  -transport of molecules  -receptors  -motors  -catalysts (enzymes)  -hormones  -structural roles  The word protein comes from the Greek πρώτα ("proteios"), meaning "of primary importance" and these molecules were first described and named by the Swedish chemist Jons Jacob Berzelius in 1838. However, proteins' central role in living organisms was not fully appreciated until 1926, when James B. Sumner showed that the enzyme urease was a protein. The first protein to be sequenced was insulin, by Frederick Sanger, who won the Nobel Prize for this achievement in 1958. The first protein structures to be solved were hemoglobin and myoglobin, by Max Perutz and Sir John Cowdery Kendrew, respectively, in 1958. Both proteins' three-dimensional structures were first determined by x-ray diffraction analysis; the structures of myoglobin and hemoglobin won Proteins are large organic compounds made of amino acids arranged in a linear chain and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. The sequence of amino acids in a protein is defined by a gene and encoded in the genetic code. Although this genetic code specifies 20 "standard" amino acids, the residues in a protein are often chemically altered in post – translational modification: either before the protein can function in the cell, or as part of control mechanisms. Proteins can also work together to achieve a particular function, and they often associate to  participate in every process within cells. Many proteins are enzymes that catalyze biochemical reactions, and are vital to metabolism. Proteins also have structural or mechanical functions, such as actin and myosin in muscle, and the proteins in the cytoskeleton, which forms a system of scaffolding that maintains cell shape. Other proteins are important in cell signalling, immune responses, cell adhesion, and the cell cycle. Protein is also necessary in animals’ diets, since they cannot synthesise all the amino acids and must obtain essential amino acids from food. Through the process of digestion, animals Protein Function Protein function is performed through two molecular events: 1) Reversible binding to a ligand - involves molecular recognition (two molecules with complementary structures) - the ligand could be any molecule (metals, organic molecules, other proteins, lipids, carbohydrates, etc…) - the ligand binds specifically to the protein binding site - binding is a bimolecular (2nd order) reaction 2) Conformational changes in the protein - proteins have dynamic structures - conformational changes are used to effect actions: - activate or inactivate proteins (allosteric transitions) PROTEIN FUNCTION: OXYGEN TRANSPORT  is mediated by proteins that bind oxygen reversibly  Myoglobin is involved in oxygen storage in peripheral tissues  Hemoglobin is involved in oxygen transport from the lungs to the peripheral tissues through blood  Oxygen binding is mediated by a prosthetic group termed Heme Why do we need oxygen transport and storage? -Oxygen has very low solubility in aqueous solvents: -Diffusion of oxygen through tissues is ineffective over long distances (> few millimeters) Gas Structure Polarity Solubiltiy in -Critical for multicellularWater (g/L) organisms Nitrogen N=N Nonpolar 0.018 (400 C) Oxygen O=O Nonpolar 0.035 (500 C) specifically bind to a certain molecule or class of molecules— they may be extremely selective in what they bind. Antibodies are an example of proteins that attach to one specific type of molecule. In fact, the enzyme-linked immunosorbent assay (ELISA), which uses antibodies, is currently one of the most sensitive tests modern medicine uses to detect various biomolecules. Probably the most important proteins, are the enzymes. These amazing molecules recognize specific reactant molecules called substrates; they then catalyze the reaction between them. By lowering the activation energy, the enzyme speeds up that reaction by a rate of 1011 or more: a reaction that would normally take over 3,000 years to complete spontaneously might take less than a second with an enzyme. The enzyme itself is not used up in the process, and is free to catalyze the same reaction with a new set of substrates. Using various modifiers, the activity of the STRUCTURE OF PROTEIN  Proteins are made of a polypeptide backbone with attached side chains.  Each type of protein differs in its amino acid sequence.  Thus the sequential position of the chemically distinct side chains gives each protein its individual properties.  The two ends of each polypeptide chain are chemically different: the end that carries the free amino group (NH3+, also written NH2) is called the amino, or N-, terminus; and the end carrying the free carboxyl group (C00–, also written COOH) is the carboxyl, or C-, terminus.  The amino acid sequence of a protein is always presented in the N to C direction, reading from left to right. Section of a protein structure showing serine and alanine residues linked together by peptide bonds. Carbons are shown in white and hydrogens are omitted for clarity. PROTEIN STRUCTURE STRUCTURE OF PROTEINS  PROTEIN STRUCTURE 1o : The linear sequence of amino acids and disulfide bonds eg. ARDV:Ala.Arg.Asp.Val. 2o : Local structures which include, folds, turns, - helices and -sheets held in place by hydrogen bonds. 3o : 3-D arrangement of all atoms in a single polypeptide chain. 4o : Arrangement of polypeptide chains into a functional protein, eg. hemoglobin. CLASSIFICATION OF PROTEINS A. Shape 1. globular – interior(non-polar grps),exterior (polar grps) 2. fibrous – strands/bundles a. Elastins & collagens b. Keratins c. myosin/muscle proteins d. fibrin B. Function 1. structural – e.g. Collagen 2. contractile proteins – e.g. Myosin & actin 3. antibodies – defense proteins 4. oxygen-binding proteins – e.g. Myoglobin 5. hormones – regulatory proteins 6. enzymes – biological catalysts 7. nutrient proteins ANALYTICAL METHODS FOR DETERMINING PROTEIN CONTENT 1. Kjeldahl method - % N x 6.25 = % protein 2. Van Slyke Method – the sample protein is treated with a strong oxidizing agent like nitrous acid, and the volume of nitrogen gas evolved is collected and measured. 3. Colorimetric determination PROTEIN DENATURATION AND RENATURATION  Denaturation of proteins involves the disruption and possible destruction of both the secondary and tertiary structures.  Since denaturation reactions are not strong enough to break the peptide bonds, the primary structure (sequence of amino acids) remains the same after a denaturation process.  Denaturation disrupts the normal alpha-helix and beta sheets in a protein and uncoils it into a random shape.   Denaturation occurs because the bonding interactions responsible for the secondary structure (hydrogen bonds to amides) and tertiary structure are disrupted.  In tertiary structure there are four types of bonding interactions between "side chains" including: hydrogen bonding, salt bridges, disulfide bonds, and non-polar hydrophobic interactions. which may be disrupted.  Therefore, a variety of reagents and conditions can cause denaturation. The most common observation in the denaturation process is the precipitation or coagulation of the protein. HEAT & UV RADIATION:  Heat can be used to disrupt hydrogen bonds and non- polar hydrophobic interactions.  This occurs because heat increases the kinetic energy and causes the molecules to vibrate so rapidly and violently that the bonds are disrupted.  The proteins in eggs denature and coagulate during cooking.  Other foods are cooked to denature the proteins to make it easier for enzymes to digest them.  Medical supplies and instruments are sterilized by heating to denature proteins in bacteria and thus destroy the bacteria. ALCOHOL DISRUPTS HYDROGEN BONDING  Hydrogen bonding occurs between amide groups in the secondary protein structure.  Hydrogen bonding between "side chains" occurs in t ertiary protein structure in a variety of amino acid combinations. All of these are disrupted by the addition of another alcohol.  A 70% alcohol solution is used as a disinfectant on the skin. This concentration of alcohol is able to penetrate the bacterial cell wall and denature the proteins and enzymes inside of the cell.  A 95% alcohol solution merely coagulates the protein on the outside of the cell wall and prevents any alcohol from entering the cell.  Alcohol denatures proteins by disrupting the side chain intramolecular hydrogen bonding. New hydrogen bonds are formed instead between the new alcohol molecule and the protein side chains.  In the prion protein, tyr 128 is hydrogen bonded to asp 178, which cause one part of the chain to be bonding with a part some distance away. After denaturation, the graphic show substantial structural changes. ACIDS AND BASES DISRUPT SALT BRIDGES:  Salt bridges result from the neutralization of an acid and amine on side chains. The final interaction is ionic between the positive ammonium group and the negative acid group. Any combination of the various acidic or amine amino acid side chains will have this effect.  As might be expected, acids and bases disrupt salt bridges held together by ionic charges. A type of double replacement reaction occurs where the positive and negative ions in the salt change partners with the positive and negative ions in the new acid or base added. This reaction occurs in the digestive system, when the acidic gastric juices cause the curdling (coagulating) of milk. HEAVY METAL SALTS:  Heavy metal salts act to denature proteins in much the same manner as acids and bases.  Heavy metal salts usually contain Hg+2, Pb+2, Ag+1 Tl+1, Cd+2 and other metals with high atomic weights.  Since salts are ionic they disrupt salt bridges in proteins. The reaction of a heavy metal salt with a protein usually leads to an insoluble metal protein salt.  This reaction is used for its disinfectant properties in external applications. For example AgNO3 is used to prevent gonorrhea infections in the eyes of new born infants. Silver nitrate is also used in the treatment of nose and throat infections, as well as to cauterize wounds.  Mercury salts administered as Mercurochrome or Merthiolate have similar properties in preventing infections in wounds.  This same reaction is used in reverse in cases of acute heavy metal poisoning. In such a situation, a person may have swallowed a significant quantity of a heavy metal salt. As an antidote, a protein such as milk or egg whites may be administered to precipitate the poisonous salt. Then an emetic is given to induce vomiting so that the precipitated metal protein is discharged from the body. HEAVY METAL SALTS DISRUPT DISULFIDE BONDS:  Heavy metals may also disrupt disulfide bonds because of their high affinity and attraction for sulfur and will also lead to the denaturation of proteins. REDUCING AGENTS DISRUPT DISULFIDE BONDS:  Disulfide bonds are formed by oxidation of the sulfhydryl groups on cysteine. . Different protein chains or loops within a single chain are held together by the strong covalent disulfide bonds. Both of these examples are exhibited by the insulin.  If oxidizing agents cause the formation of a disulfide bond, then reducing agents, of course, act on any disulfide bonds to split it apart. Reducing agents add hydrogen atoms to make the thiol group, -SH. The reaction is: REVERSIBLE DENATURATION ENZYMES Enzymes are proteins which act as biological catalysts. Over 1500 have been isolated. Human genome project scientists estimate that there are about 30,000 (>100,000) enzymes in a human. Active (catalytic) site is a crevice which binds a substrate. Lock & key metaphore....but, protein can change conformation. The active site is evolutionarily conserved. NOMENCLATURE  Typically add “-ase” to name of substrate  e.g. lactase breaks down lactose (dissacharide of glucose and galactose)  IUBMB classifies enzymes based upon the class of organic chemical reaction catalyzed:  1) oxidoreductase - catalyze redox reactions  dehydrogenases, oxidases, peroxidases, reductases  2) transferases - catalyze group transfer reactions; often require coenzymes  3) hydrolases - catalyze hydrolysis reactions  4) lyases - lysis of substrate; produce contains double bond  5) isomerases - catalyze structural changes; isomerization 6) ligases - ligation or joining of two substrates with input of energy, usually from ATP hydrolysis; often called synthetases or synthases SPECIFICITY OF ENZYMES  One of the properties of enzymes that makes them so important as diagnostic and research tools is the specificity they exhibit relative to the reactions they catalyze.  A few enzymes exhibit absolute specificity; that is, they will catalyze only one particular reaction. Other enzymes will be specific for a particular type of chemical bond or functional group. In general, there are four distinct types of specificity: 1. Absolute specificity - the enzyme will catalyze only one reaction. 2. Group specificity - the enzyme will act only on molecules that have specific functional groups, such as amino, phosphate and methyl groups. 3. Linkage specificity - the enzyme will act on a particular type of chemical bond regardless of the rest of the molecular structure. 4. Stereochemical specificity - the enzyme will act on a particular steric or optical isomer. FACTORS THAT AFFECT THE RATE OF ENZYME ACTION ENZYME CONCENTRATION  In order to study the effect of increasing the enzyme concentration upon the reaction rate, the substrate must be present in an excess amount; i.e., the reaction must be independent of the substrate concentration.  Any change in the amount of product formed over a specified period of time will be dependent upon the level of enzyme present. Graphically this can be represented as: SUBSTRATE CONCENTRATION  It has been shown experimentally that if the amount of the enzyme is kept constant and the substrate concentration is then gradually increased, the reaction velocity will increase until it reaches a maximum.  After this point, increases in substrate concentration will not increase the velocity (delta A/delta T). TEMPERATURE EFFECTS  Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the temperature is raised.  A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%. Variations in reaction temperature as small as 1 or 2 degrees may introduce changes of 10 to 20% in the results.  In the case of enzymatic reactions, this is complicated by the fact that many enzymes are adversely affected by high temperatures. As shown in Figure 13, the reaction rate increases with temperature to a maximum level, then abruptly declines with further increase of temperature. Because most animal enzymes rapidly become denatured at temperatures above 40°C, most enzyme determinations are carried out somewhat below that temperature.  Over a period of time, enzymes will be deactivated at even moderate temperatures. Storage of enzymes at 5°C or below is generally the most suitable. Some enzymes lose their activity when frozen. EFFECTS OF PH  Enzymes are affected by changes in pH. The most favorable pH value - the point where the enzyme is most active - is known as the optimum pH.  Extremely high or low pH values generally result in complete loss of activity for most enzymes. pH is also a factor in the stability of enzymes. As with activity, for each enzyme there is also a region of pH optimal stability. EFFECTS OF INHIBITORS ON ENZYME ACTIVITY  Enzyme inhibitors are substances which alter the catalytic action of the enzyme and consequently slow down, or in some cases, stop catalysis.  There are three common types of enzyme inhibition - competitive, non-competitive and substrate inhibition.  Most theories concerning inhibition mechanisms are based on the existence of the enzyme- substrate complex ES. As mentioned earlier, the existence of temporary ES structures has been verified in the laboratory. MECHANISM OF INHIBITION 1. Competitive inhibition occurs when the substrate and a substance resembling the substrate are both added to the enzyme.  A theory called the "lock-key theory" of enzyme catalysts can be used to explain why inhibition occurs. 2. Non-competitive inhibitors are considered to be substances which when added to the enzyme alter the enzyme in a way that it cannot accept the substrate.  3. Substrate inhibition will sometimes occur when excessive amounts of substrate are present.  Additional amounts of substrate added to the reaction mixture after this point actually decrease the reaction rate.  This is thought to be due to the fact that there are so many substrate molecules competing for the active sites on the enzyme surfaces that they block the sites (Figure 12) and prevent any other substrate molecules from occupying them.