Protein Structure and Function PDF

Amino Acids Structure and Function Proteins ❖ They are the most abundant intracellular macromolecule – most of the protein mass is found in the skeletal muscle ❖ The molecule is comprised of the elements carbon (C), hydrogen (H), oxygen (O), nitrogen (N) and small quantities of sulphur (S) – CHO...

Amino Acids Structure and Function Proteins ❖ They are the most abundant intracellular macromolecule – most of the protein mass is found in the skeletal muscle ❖ The molecule is comprised of the elements carbon (C), hydrogen (H), oxygen (O), nitrogen (N) and small quantities of sulphur (S) – CHONs ❖ Proteins found in animal contains 0.5 -2.0% sulphur except for insulin - 3.4% ❖ They are biological catalysts – enzymes ❖ They act as carrier molecules transporting small molecules and ions e.g. haemoglobin (protein) transports oxygen in the erythrocytes ❖ They responsible for the high tensile strength of the skin and bone ❖ They are responsible for immunoregulation ❖ Receptor proteins help in the transmission of nerve impulses ❖ They are a major component of muscle Amino Acids ❖ Proteins are made up of amino acids linked together by peptide bonds ❖ They contain both an amino (-NH2) and a carboxylic acid (-COOH) functional group ❖ There is also a H atom and a R group which represents either H or a hydrocarbon chain ❖ All are bonded to an α – carbon, which is also a chiral carbon ❖ Amino acids are dipolar ions (zwitterions) in solution ❖ In the dipolar form amino group becomes positively charged (NH3+) and the carboxylic acid becomes negatively charged (COO-) http://cyberlab.lh1.ku.ac.th/elearn/faculty/veterin/vet69/Biochemistry%20Web%20Job/amino%20and% 20protein/zwitterion2.jpg Amino Acids ❖ There are 20 amino acids that can make up proteins ❖ 9 essential amino acids ❖ 11 non essential amino acids ❖ Essential amino acids – are not synthesized in the body and are normally required in the diet ❖ Non essential amino acids –are synthesized de novo in humans Amino Acids ❖ Amino acids can be grouped based on the properties of the R group non polar polar uncharged acidic (negatively charged) basic (positively charged) Peptide Bond Formation ❖ Amino acids can undergo condensation reactions to form peptide bonds ❖ When 2 amino acids react → dipeptide ❖ 3 amino acids → tripeptide ❖ < 10 amino acids - oligopeptide ❖ more than 20 amino acids → polypeptide ❖ Polypeptide with a MW > 6000 or ≥ 100 amino acids → protein ❖ Each amino acid in a polypeptide chain is termed a residue ❖ The two ends of the chain are named amino acid terminal (N- terminal) and carboxylic terminal (C-terminal) respectively ❖ These terminals are the only ionizable groups (except for the side chain) in a protein Biological Roles of Peptides ❖ Intermediates for the formation of proteins ❖ Antibacterial properties e.g. penicillin ❖ Growth factors e.g. folic acid ❖ Hormones e.g. insulin ❖ Control oxidation-reduction potential of cell e.g. glutathione ❖ Increased levels in urine may be indicative of psychological /neurological disorders such as depression and schizophrenia (Gluten/casein peptides) Glutathione ❖ Natural antioxidant ❖ Glutathione is produced in cells as needed. It is used as an antioxidant to prevent damage to cells from heavy metals, free radicals and peroxides ❖ It is made from γ-glutamic acid – cysteine – glycine, which is also written: γ-Glu – Cys – Gly http://guweb2.gonzaga.edu/faculty/cronk/biochem/images/glutathione.gif Angiotensin ❖ Angiotensin is a potent vasoconstrictor that is synthesized by cells ❖ Angiotensin I is a decapeptide. It is biologically inactive and acts solely as a precursor for angiotensin II ❖ Angiotensin II is an octapeptide and causes blood vessels to constrict when there is a cut (to stop the bleeding) ❖ Angiotensin I is converted to angiotensin II when needed by the angiotensin converting enzyme – ACE Aspartame  Artificial non saccharide sweetener  200 times sweeter than sugar  Claimed to be linked to cancer, alzheimer, lupus, headache, Parkinsons diesease Oxytoxin: Love Hormone  Neuropeptide Regulates social interaction and sexual reproduction It acts as buffer against stress, depression and anxiety Hydrolysis of Proteins ❖ Proteins can be hydrolysed (using acid or a suitable enzyme) to form individual amino acids http://www.chemguide.co.uk/organicprops/aminoacids/hydrolprotein.gif Protein Structure ❖ Protein structure can be grouped into four levels of organization 1. Primary structure – linear sequence of amino acids 2. Secondary structure – spatial arrangement of a portion of the polypeptide. Three arrangements exist i.e. (a) α- helix, (b) β- pleated sheets, (c) β- bends 3. Tertiary structure – describe the shape of the entire polypeptide 4. Quaternary structure – spatial arrangement of many polypeptide chains Protein Structure ❖ Protein structure can be grouped into four levels of organization 1. Primary structure – linear sequence of amino acids. Main linkage – peptide bonds How important is the primary sequence? ❖ Some small change in a.a can make a big difference ❖ Normal Hb has 2 alpha chains and 2 beta chains. Change in the a.a. sequence of the beta chain will result in abnormal Hb Normal Hb - Thr-Pro-Glu-Glu-Lys-Ala Sickle cell Hb - Thr-Pro-Val-Glu-Lys-Ala ❖ Red blood cells carrying HbS behave normally when there is an ample supply of oxygen. Reduced oxygen concentration results in the rbc becoming sickle-shaped ❖ Sickled cells may clog capillaries ❖ The body’s defense mechanisms destroy the clogging cells and the loss of blood cells causes anaemia 2. Secondary structure – spatial arrangement of a portion of the polypeptide. Main linkage- H bonding (a) α- helix e.g. α-keratin (b) β- pleated sheets e.g. silk fibroin (c) β- bends α-keratin Silk fibroin Protein Structure 3. Tertiary structure – describe the shape of the entire polypeptide…folding of the helices of globular proteins. All the chemical bonds are found in the structure e.g. myoglobin (cardiac and red skeletal muscle) Chaperones – a protein that helps other proteins to fold into the biologically active conformation and enables partially denatured proteins to regain their biologically active conformation 4. Quaternary structure – spatial arrangement of many polypeptide subunits…all chemical bonds are found in the structure e.g. haemoglobin (tetrameric protein) Nature of Enzymes ❖ They are proteins ❖ Biological catalysts…Unlike inorganic catalysts (a) enzymes do not last indefinitely (b) They are biological products ❖ They speed up biochemical reactions by lowering the activation energy necessary for a reaction to take place ❖ Enzymes can be denatured and precipitated with salts, solvents and other reagents. They have molecular weights ranging from 10,000 to 2,000,000 http://g11-bioa-2011-12.wikispaces.com/file/view/ch06c1.jpg/261340130/ch06c1.jpg Enzymes ❖ Their reactions may be anabolic - involved in synthesis or catabolic – involved in breakdown ❖ The name of the enzyme always end with the suffix –ase ❖ Enzymes bind to reactants (substrate) ❖ The area on the enzyme that binds to the substrate is called the active site ❖ The active site and the substrate have complementary shape enabling them to bind together (like pieces of a jig saw puzzle) ❖ The interaction is not rigid as would occur between a lock and a key (lock and key hypothesis ) Enzymes ❖ The enzyme has an internal flexibility and is able to undergo conformational changes, adjusting to the shape of the substrate (induced fit hypothesis) http://image.tutorvista.com/content/surface-chemistry/substrates-enzymes-induced-fit-model.jpeg Enzymes ❖ Enzymes are therefore specific for a particular substrate ❖ When an enzyme-substrate is formed it is activated to form products ❖ The products formed cannot fit into the active site and escapes leaving the active site of the enzyme free ❖ Many enzymes require non protein components to carry out their functions (cofactors) ❖ The enzyme-cofactor complex is called a haloenzyme ❖ Enzyme portion without the cofactor is called an apoenzyme Enzymes ❖ There are three groups of cofactors 1. Inorganic ions - eg. Salivary amylase activity is increased in the presence of chloride ions (Cl-) 2. Prosthetic groups – eg. Flavin adenine dinucleotide (FAD), haem and biotin 3. Coenzymes – eg. Adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD [oxidised form], NADH [reduced form]) Enzyme Cofactors  A simple enzyme is an active enzyme that consists only of protein  Many enzymes are active only when they combine with cofactors such as metal ions or small molecules Coenzymes  A coenzyme is a cofactor that is a small organic molecule such as a vitamin  In the active site, activity may be assisted by organic coenzymes  A coenzyme prepares the active site for catalytic activity  Coenzymes: some vitamins, e.g.. B complex and Vit C  Apoenzyme is inactive, add the cofactor, makes holoenzyme which is active  They may contribute to the enzyme action by supplying oxidation or reduction action (for example NAD+ takes up H2 to make NADH/H+) FAD also, goes to FADH2 30 Metal Ions  Metal ions are needed in some enzyme catalysis, for example: iron, zinc, copper  Many active enzymes require a metal ion  Zn2+, a cofactor for carboxypeptidase, stabilizes the carbonyl oxygen during the hydrolysis of a peptide bond Some enzymes and their Metal Ion Cofactors Metal ion cofactor Function Enzyme Fe2+/Fe3+ Oxidation-reduction Oxidation-reduction Catalase Cytochrome oxidase Zn2+ Used with NAD+ Mg2+ Hydrolyses phosphate esters Alcohol dehydrogenase Carbonic anhydrase Carboxypeptidase A Glucose-6-phosphatase Mn2+ Ni2+ Removes electrons Hydrolyses amides Arginase Urease Enzymes http://academic.pgcc.edu/~kroberts/Lecture/Chapter%205/05-04_Holoenzyme_L.jpg Diagnostic Enzymes  Diagnostic enzymes are used for clinical diagnosis  The level of diagnostic enzyme determines the amount of damage in tissues. Condition Diagnostic enzymes elevated Heart attack, or liver disease (cirrhosis, hepatitis) Lactate dehydrogenase (LDH) Aspartate aminotransferase (AST) Heart attack Creatine kinase (CK) Hepatitis Alanine transaminase (ALT) Liver (carcinoma) or bone disease Alkaline phosphatase (ALP) Pancreatic disease Amylase Lipase (more specific) Prostate carcioma Acid phosphatase (ACP) Therapeutic Applications of Enzymes  Enzymes are used for the treatment of a broad variety of diseases by virtue of the fact that they catalyze chemical reactions with great speed and specificity  There are different categories of therapeutic enzymes. They include: Oncology – Enzymes are used to control the growth of select neoplasms or leukemias Bacteria asparaginase has been used for the treatment of leukaemia Anticoagulants - Fibrinolytic drugs act on fibrinogen, a plasma glycoprotein that is converted into fibrin during clot formation. These include streptokinase, urokinase, alteplase, reteplase, tenecteplase, and tissue plasminogen activator [t-PA] Thrombolytic - capable of rapidly lysing the clots which cause, or contribute to, myocardial infarction, phlebitis, pulmonary embolisms, occluded catheters, and allied conditions e.g. Urokinase and Streptokinase are basically used in the myocardial infarction which dissolves purulent material or the clot Therapeutic Applications of Enzymes  Replacement Therapy - parenteral replacement therapy for genetic diseases attributable to a deficiency of a specific enzyme or family of enzymes e.g. prolactazyme is a proenzyme that produces lactase in the stomach (lactose intolerance), Aglucerase – used in the treatment of Gaucher’s disease  Antidotes to poisons or as counteragents capable of mitigating the delirious effects of toxins - Enzyme Inhibition  An enzyme inhibitor is any substance that can decrease the rate of an enzyme catalyzed reaction  Enzyme inhibition can be reversible or irreversible (a) Reversible inhibition: an inhibitor binds reversibly to an enzyme. An equilibrium is established and at some point in time the inhibitor is removed from the enzyme. (b) Irreversible Inhibition: an inhibitor forms a covalent bond with specific functional group of the enzyme and renders the enzyme inactive. Irreversible inhibition essentially destroys the enzyme so that it can no longer function as an enzyme.  Enzymes and substrates bind together, a substance that copies the substrate may be able to bind in its place. This is called competitive inhibition  A substance may bind in some other place on enzyme this is called allosteric inhibition 38  Allosteric inhibition is also called non-competitive Enzyme Inhibition  Enzyme inhibition is also used for medicinal therapy  Many medicines are enzyme inhibitors, look up examples 39 Competitive Inhibitors  The inhibitor has a similar shape to the usual substrate for the enzyme, and competes with it for the active site  However, once it is attached to the active site, no reaction takes place Normally With inhibitor Competitive Inhibitors Competitive Inhibitors ❖ The complex does not react any further to form products - but its formation is still reversible. It breaks up again to form the enzyme and the inhibitor molecule. ❖ If you increase the concentration of the substrate, then the substrate can out-compete the inhibitor, and so the normal reaction can take place at a reasonable rate. ❖ Methanol poisoning occurs because methanol is oxidized to formaldehyde and formic acid which attack the optic nerve causing blindness. Ethanol is given as an antidote for methanol poisoning because ethanol competitively inhibits the oxidation of methanol. Ethanol is oxidized in preference to methanol and consequently, the oxidation of methanol is slowed down so that the toxic by-products do not have a chance to accumulate. Non-competitive Inhibitors/ Allosteric Inhibitors ❖ A non-competitive inhibitor does not attach itself to the active site, but attaches somewhere else on the enzyme ❖ By attaching somewhere else it affects the structure of the enzyme (conformational change at the active site) and so the way the enzyme works ❖ Because there is no competition involved between the inhibitor and the substrate, increasing the substrate concentration will not help ❖ Some non-competitive inhibitors attach irreversibly to the enzyme, and therefore stop the enzyme from working permanently. Others attach reversibly Non-competitive inhibitors ❖ Since many enzymes contain sulfurhydrl (-SH), alcohol, or acid groups as part of their active sites, any chemical which can react with them acts as an irreversible inhibitor ❖ Heavy metals such as Ag+, Hg2+, Pb2+ have strong affinities for -SH groups. Mercury (Hg) and Lead (Pb) poisoning can cause permanent neurological damage ❖ Heavy metal poisoning is treated by administering chelating agents e.g. ethylenediaminetetraacetic acid (EDTA) Uncompetitive Inhibition  Uncompetitive inhibitors bind only to the enzyme-substrate complex (ES) forming an unreactive enzyme-inhibitor-substrate complex (EIS)  Can only bind if the substrate has bound  This type of inhibition is reversible with decreasing [S] Medicinal Inhibitors  HIV/AIDS drug Ritonavir is a protease inhibitor and inhibits an essential protein breakdown; reduces the virus count (used in combination with other medication)  Orlistat is a lipase inhibitor that reduces the amount of fat that the body absorbs  Captopril inhibits angiotensin converting enzyme (ACE) and that lowers blood pressure  Sulfanilamide kills bacteria by inhibiting bacterial enzymes as do the penicillin  Some chemotherapeutic medicines inhibit production of bases, for example 5- fluorouracil which inhibits thymidine production in cancer cell growth  Viagra inhibits an enzyme that degrades cGMP so that cGMP lasts longer to allow blood flow and so prolongs erection  Acyclovir, which inhibits the enzymes that copy viral DNA to combat herpes. 47 Regulation of Enzymes  Enzyme inhibition can regulate a metabolic process to stop making products – this is called feedback inhibition. End Product Inhibition/ Negative Feedback Consider the series of enzyme catalyzed reaction A→B→ C→ D ❖ If excess D (end-product) is formed, it non-competitively inhibits the enzyme that converts A to B (must be a non-competitive inhibitor otherwise the system wouldn’t work at high concentrations of A). This effectively stops the production of B, and thus C and D. ❖ As no more D is being made, the excess D will eventually be used up. When this happens, the inhibition on the A → B reaction is lifted, and the system starts up again ❖ This is an example of negative feedback and is useful in ensuring that endless quantities of unnecessary end product are not produced Factors Affecting Enzyme Kinetics ❖ The activity of an enzyme is affected by its environmental conditions ❖ Changing these alter the rate of reaction caused by the enzyme ❖ In nature, organisms adjust the conditions of their enzymes to produce an Optimum rate of reaction, where necessary, or they may have enzymes which are adapted to function well in extreme conditions where they live Factors affecting Enzyme Kinetics: Effect of Temperature ❖ Little activity at low temperature ❖ Rate increases with temperature ❖ Enzymes are most active at optimum temperatures (usually 37°C in humans) ❖ Increasing temperature increases the Kinetic Energy that molecules possess ❖ Since enzymes catalyse reactions involves enzymes randomly colliding with Substrate molecules, increasing temperature increases the rate of reaction, forming more products ❖ However, increasing temperature also increases the movement of enzyme molecules. This puts strain on the bonds that hold them together ❖ As temperature increases, more bonds, especially the Hydrogen and Ionic bonds, will break as a result of this strain ❖ Breaking bonds within the enzyme will cause the active site to change shape ❖ This change in shape means that the active site is less complementary to the shape of the substrate, so that it is less likely to catalyse the reaction. Eventually, the enzyme will become denatured and will no longer function Factors affecting Enzyme Kinetics: Effect of pH ❖ Enzyme works best within a narrow pH range ❖ Each enzyme works best at particular pH, known as its optimum pH level ❖ At extreme pH levels, enzymes lose their shape and function and become denatured ❖ H+ and OH- ions are charged and therefore interfere with Hydrogen and Ionic bonds that hold the enzyme, since they will be attracted or repelled by the charges created by the bonds. This interference causes a change in shape of the enzyme, and importantly, its active site ❖ Any change in pH above or below the optimum will quickly cause a decrease in the rate of reaction, since more of the enzyme molecules will have active sites whose shapes are not, or at least less, complementary to the shape of their substrate ❖ Small changes in pH above or below the optimum do not cause a permanent change to the enzyme, since the bonds can be reformed. However, extreme changes in pH can cause enzymes to denature and permanently lose their function Optimum pH Levels  Most enzymes of the body have an optimum pH of about 7.4  In certain organs, enzymes operate at lower and higher optimum pH values Enzyme Location Substrate Optimum pH Pepsin Stomach Peptide bonds 2 Urease Liver Urea 5 Sucrase Small intestine Sucrose 6.2 Pancreatic amylase Pancreas Amylase 7 Trypsin Small intestine Peptide bonds 8 Arginase Liver Arginine 9.7 Factors Affecting Enzyme Kinetics: Effect of concentration ❖ Changing the Enzyme and Substrate concentrations affect the rate of reaction of an enzyme catalysed reaction ❖ Controlling these factors in a cell is one way that an organism regulates its enzyme activity and so its metabolism ❖ Changing the concentration of a substance only affects the rate of reaction if it is the limiting factor: that is, it the factor that is stopping a reaction from preceding at a higher rate ❖ (a) Substrate Concentration ❖ Increasing substrate concentration increases the rate of reaction. This is because more substrate molecules will be colliding with enzyme molecules, so more product will be formed ❖ However, after a certain concentration, any increase will have no effect on the rate of reaction, since substrate concentration will no longer be the limiting factor ❖ The enzymes will effectively become saturated, and will be working at their maximum possible rate (b) Enzyme Concentration ❖ Increasing Enzyme Concentration will increase the rate of reaction, as more enzymes will be colliding with substrate molecules ❖ However there will be no effect when the enzyme concentration is now the limiting factor