PRELIM TOPICS Pharmaceutical Biochemistry -92-253.pdf

Protein Hydrolysis Reverse of peptide bond formation Results in the regeneration of an amine and carboxylic acid functional groups Protein digestion - Enzyme-catalyzed hydrolysis Free amino acids produced are absorbed into the bloodstream and transported to the liver f...

Protein Hydrolysis Reverse of peptide bond formation Results in the regeneration of an amine and carboxylic acid functional groups Protein digestion - Enzyme-catalyzed hydrolysis Free amino acids produced are absorbed into the bloodstream and transported to the liver for the synthesis of new proteins Hydrolysis of cellular proteins to amino acids is an ongoing process, as the body resynthesizes needed molecules and tissue Protein Hydrolysis Complete hydrolysis All peptide bonds are broken freeing all AAs; products are free AAs Partial hydrolysis Some peptide bonds are broken; products are free AAs and small peptides Protein Hydrolysis Protein digestion is simply enzyme-catalyzed hydrolysis of ingested proteins The free amino acids produced from this process are absorbed through the intestinal wall into the bloodstream and transported to the liver In the liver, they become raw materials for the synthesis of new protein tissue Also, the hydrolysis of cellular protein to amino acids is an ongoing process, as the body re-synthesizes needed molecules and tissue. Protein Denaturation Protein Denaturation Partial or complete disorganization of a protein’s characteristic 3D shape (disordered) Loses biochemical activity Does not affect primary structure of proteins Protein Denaturation Sometimes denaturation can be reversed = renaturation (“refolded”), but usually is irreversible Loses water solubility – usual property of denatured proteins Precipitation of denatured proteins = coagulation Protein Denaturation Applications “Cooked” proteins are easily digested because it is easier for digestive enzymes to act on denatured proteins Cooking also kills microorganisms also through protein denaturation Ham & bacon harbors parasites that can cause trichinosis Protein Denaturation Applications Cauterization = In surgery, heat is used to seal small blood vessels Heating of surgical instruments for sterilization Patient’s body temperature should not exceed 106°F(41°C) as body enzymes begin to inactive lethal effects UV from sun causes denaturation (sunburn) Protein Denaturation RENATURATION o Restoration process in which protein is “refolded” o Only very few proteins undergo renaturation. o Denaturation is irreversible in most proteins. Two Types of Denaturing Agents: o Physical: heat, high pressure, UV rays, agitation o Chemical: acids, bases, organic solvents salts of heavy metals Consequences of Denaturation o Physical: decreased solubility; precipitation o Chemical: increased viscosity o Biological: loss of hormonal and/or enzymatic activity Classification of Proteins Classification of Proteins According to Composition/ Hydrolysates: Simple Proteins -Proteins that yields only amino acids on hydrolysis Classification of Proteins According to Composition/ Hydrolysates: Simple Proteins According to Composition/ Hydrolysates: Conjugated Proteins -those on hydrolysis will yield non-protein substances (prosthetic groups) in addition to amino acids According to Composition/ Hydrolysates: Derived Proteins Substances produced by the action of heat, acids, alkalis, or enzymes on simple or conjugated proteins; includes hydrolytic products of the original protein According to Structure  According to Biological Roles 1. Structural protein Serve as supporting filaments, cables with sheets to give biological structures strength and protection a. Collagen:fibrous protein found in tendons and cartilage with high tensile strength o leather: almost pure collagen b. Elastin: structural protein capable of stretching in two dimensions o ligaments c. Keratin: largely tough insoluble protein found in hair, fingernails and feathers d. Fiborin: found in silk fibers and spider webs  According to Biological Roles 1.Structural protein e. Resilin: has perfect elastic property found in wing hinges of some insects f. Glycoproteins: acts as cell membrane and cell coats g.Mucoprotein: mucous membrane, synovial fluids h.Sclerotin: exoskeleton of insects  According to Biological Roles 2. Catalytic Protein/Enzymes Most varied and most highly specialized proteins with catalytic activity Serves to increase rate of thousands of chemical reactions Biochemical reactions involve exchange of electrons between atoms of reacting molecules which is being hastened by the presence of enzymes o Enzymes are protein catalysts, capable of enhancing the rates of reactions by factors of up to 1014. For example, the enzyme carbonic anhydrase catalyzes the reaction: CO2 + H2O ↔H+ + HCO3- by a factor of 107 over the uncatalyzed reaction.  According to Biological Roles 2. Catalytic Protein/Enzymes o Alcohol Dehydrogenase: important in alcohol fermentation o Arginase: hydrolyzes arginine o Ribonuclease: hydrolyzes RNA o Urease: hydrolyzes urea to form CO2 + H2O o Cytochrome C: responsible for the transport electrons o Chymotrypsin: cleaves the polypeptide chain from the C terminal of Arginine and Lysine o Trypsin: cleaves the polypeptide chain from the N terminal of Arginine and Lysine o Rennin: digestive enzyme which acts on casein o Aminopolypeptidase: sequentially cleaves the protein chain from the N terminal  According to Biological Roles 3. Nutrient and Storage proteins Seeds of many plants store nutrients (proteins required for growth of the germinating seedling) Eg., seed proteins of wheat, corn and rice a. Ovalbumin: major protein in egg white b. Casein: major protein in milk c. Ferritin: stores iron in the spleen d. Gliadin: seed storage in wheat e. Zein: seed storage in corn f. Thymus histone: stored in thymus gland  According to Biological Roles 4. Transport proteins These bind and carry specific molecules and ions from one organ to another Many small molecules are transported both inside and outside cells when bound to carrier proteins. Examples include the oxygen transport and storage proteins hemoglobin and myoglobin, respectively. The proteins in membranes often permit the passage of both small and large molecules through membranes.  According to Biological Roles 4. Transport proteins Binds to and carries specific molecules or ions from place to place o Hemoglobin: carry oxygen from lungs to body organs o Β1-lipoproteins: carry lipids from the liver to the other organs o Hemocyanin: transport oxygen in the blood of some invertebrates o Myoglobin: transport oxygen in the muscles o Serum albumin: transports fatty acids o Transportin: transports steroids o Transcobalamin: transports vitamin B12 o Transferrin/Siderophilin/Iron-Binding Globulin: transports iron o Cytochrome C: transports electrons o Ceruloplasmin: transports copper in the blood  According to Biological Roles 5. Contractile proteins Provides cells and organelles with the ability to contract, to change shape and to move about o Actin: thin and moving filaments of the myofibril in skeletal muscles; o Myosin: thick and stationary filament in skeletal muscles; moving filaments of the myofibril o Tribulin: protein from which microtubules are built; they act in concert with other proteins in flagella and cilia to propel cells. o Kinesin moves protein cargoes around cells along microtubule “rails” formed by tubulin, which is also present in the flagella of sperm cells.  According to Biological Roles 6. Toxins Clostridium botulinum: causes bacterial food poisoning Diphtheria toxin: bacterial toxin Snake venoms: contains enzymes which hydrolyze phosphoglycerides Ricin: toxic protein of castor bean  According to Biological Roles 7. Regulatory Proteins: help regulate cellular and physiological activity Hormones: product of living cells that circulates in body fluids or sap and produces a specific effect (stimulatory) on the activity of cells remote from its point of origin o Insulin: regulates blood glucose level o Growth hormones: control growth of bones o Adrenocorticotrophic hormone: regulated corticosteroid synthesis  According to Biological Roles 8. Antibody/Protective proteins Defend organisms against invasion by other species and protect them from injury o Immunoglobulins/Antibodies ▪ Specialized proteins made by the lymphocytes of vertebrates ▪ Released by plasma cells to fight infection ▪ Recognize and inactivate invading bacteria ▪ Can recognize and precipitate or neutralize invading bacteria, viruses, or foreign protein from other species ✔Macroglobulin (IgM): natural antibody, first to fight infections but is short-lived for it cannot pass through the placenta ✔Classic Antibody (IgG): takes over the IgM in continued infection ✔Immunosurface protection antibody (IgA): present in all body secretions most especially breast milk ✔Regain/Homocytotrophic Antibody (IgE): increased during allergic reactions like asthma, pneumonia, hay fever ✔IgD: structure and function still unknown  According to Biological Roles 8. Antibody/Protective proteins o Complement: forms complexes with some antigen-antibody systems o Fibrinogen: precursor of fibrin in blood clotting o Thrombin: component of the clotting mechanism Diseases Associated with Proteins COLLAGEN Found in skin, bones, tendon, cartilage and teeth Most abundant protein in mammals Water-insoluble fibers Has great tensile strength Function: stress-bearing of connective tissues  COLLAGEN STRUCTURE Contains repeating X-Y-Gly (where X is any amino acid; Y is often Pro or hyroxyproline); Lys, 5-hydroxylysine and His residues are present at some of the X & Y in the triplet repeat Has unusual amino acids: 4-hydroxyproline 3-hydroxyproline 5-hydroxylysine Pro and HyPro make up 30% of residues Each polypeptide chain has about 1,000 AA residues  COLLAGEN DISEASES Osteogenesis imperfecta and Ehlers- Danlos Syndrome are caused by mutant alleles of collagen genes. Results from replacement of Gly residues with AA with large R groups such as Cys and Ser = disruption of structure and Osteogenesis imperfecta function. Ehlers-Danlos Syndrome  COLLAGEN DISEASES In the synthesis of collagen, Vitamin C is required as cofactor for enzymes prolyl hydroxylase and lysyl hydroxylase to function. These enzymes are responsible for the hydroxylation of Pro and Lys in collagen. Impaired hydroxylation of Pro and Lys results to collagen instability and the connective tissue problem seen in scurvy (deficiency in Vit C).  ELASTIN A protein that gives connective tissue its elastic properties-like rubber band. Found in lungs, walls of large blood vessels and elastic ligaments Skin to allow skin to stretch and then spring back to shape. Vessels and heart to stretch to control blood pressure. In joints to allow cartilage to absorb shock and avoid injury.  ELASTIN Consists predominantly of nonpolar AA residues 1/3 Gly, 1/3 Val + Ala, rich in Pro and produces a random coil conformation Elastin fibers associate by desmosine crosslinks (formed by 3 modified Lys and 1 Lys residues) Can be stretched to several times their normal length but recoil to original shape when relaxed. Desmosine ELASTIN DISEASES Deletion of the elastin gene was found in approx. 90% of patients with Williams Syndrome  MYOGLOBIN Made up of a single polypeptide chain with 153 AA; 8 alpha-helices; globular protein Has a single heme group Has high affinity for oxygen Primary function: oxygen storage protein, abundant in skeletal muscles  HEMOGLOBIN Tetrameric protein made up of 2 α and 2 β subunits both with 153 AAs. Each monomer is similar to myoglobin structure. Each monomer has 1 heme. Thus can bind up to 4 oxygen molecules. Comparison between myoglobin and hemoglobin. Primary function: Oxygen transport protein  HEMOGLOBIN DISEASE SICKLE CELL ANEMIA Sickle or crescent shape RBC Substitution of Val to Glu on the 6th aa of β subunit Change in the 10 structure produce hydrophobic patch on the surface of Hb hydrophobic patch interacts with other hydrophobic patches causing the Hb to aggregate into strands that align into insoluble fibers Less efficient in delivering O2  INSULIN Produced from β-cells of Islets of Langerhans in the pancreas Made up of 2 polypeptide chains (51 AAs); with inter- and intra-chain disulfide linkage It promotes glucose intake from blood into fat, liver and skeletal muscle cells. Hypoglycemic hormone Glucose in the cell is converted to glycogen (glycogenesis) or fats (lipogenesis).  GLUCAGON Produced from α-cells of Islets of Langerhans in the pancreas Made up of single polypeptide chain (29 AAs) It elevates the blood glucose level by promoting synthesis of glucose (gluconeogenesis) and breakdown of glycogen into glucose molecules (glycogenolysis). Hyperglycemic hormone  INSULIN DISEASE DIABETES MELLITUS Type 1: failure of the pancreas to produce enough insulin Type 2: a condition in which cells fail to respond to insulin (insulin resistance)  ANTIBODY Also known as immunoglobulins (Ig) Secreted mostly by the differentiated B lympocytes (plasma cells). Ig is a glycoprotein. Y-shaped molecule consists of 2 identical Light chains (~25kDa) and 2 identical Heavy chains (~50kDa) held together by disulfide bonds. Each chain contains constant and variable regions. - The variable regions in Ab serve as binding sites to the epitope of the antigen.  ANTIBODY ISOTYPES IgA – found in mucosal areas, such as the gut, respiratory tract and urogenital tract. Prevents colonization by pathogens. Also found in saliva, tears and breast milk. IgD – found mainly as antigen receptor on B cells that have not been exposed to antigens. It has been shown to activate basophils and mast cells to produce antimicrobial factors. IgE – binds to allergens and triggers histamine release from mast cells and basophils, involved in allergy. Also protects against parasitic worms.  ANTIBODY ISOTYPES IgG – has 4 forms. Provides the majority of antibody-based immunity against invading pathogens. This is the only Ig that can cross the placenta to give passive immunity to the fetus. IgM – expressed on the surface of B cells (monomer) and in a secreted form (pentamer) with very high avidity. Eliminates pathogens in the early stages of B cell-mediated (humoral) immunity before there is sufficient IgG.  MECHANISM OF ANTIBODY REACTIONS Can distinguish “self” from “non-self” molecules. Ab can tag a microbe or an infected cell for attack by other parts of the immune system, or Ab can neutralize its target directly (for example, by blocking a part of a microbe that is essential for its invasion and survival). Auto-immune disease – presence of self- reactive immune response. Body “fights” against self. AMINO ACIDS AND PEPTIDES Pharmaceutical Biochemistry Lecture  OUTLINE OF DISCUSSION 1. Biochemical functions of protein systems 2. Amino acid structure and classification 3. Amino acid interactions 1. Acid-base behavior of amino acids 2. Oxidation 4. Peptide bond formation, structural features and stability 5. ipH (or pI) calculation of amino acids and peptides. PROTEINS Naturally occurring, unbranched polymers in which the monomer units are amino acids. The 2nd most abundant substance in nearly all cells (15% of a cell’s overall mass). Contain C, H, O, N, S and sometimes contain Fe, P and some metals (specialized proteins). PROTEINS Peptides in which at least 40 amino acids are present. — Several proteins with >10,000 AA residues are known — Common proteins have 400-500 AA residues — Small proteins have 40-100 AA residues More than one polypeptide chain may be present in a protein — Monomeric (one polypeptide chain) — Multimeric (2 or more polypeptide chain) BIOCHEMICAL FUNCTIONS OF PROTEINS Structural – for supporting of structures and tissues - e.g. collagen (bones and skin), elastin (skin) Catalytic – for hastening biochemical reactions — e.g. amylase (saliva), lipase (pancreas) Transport – for transport of other substances — e.g. hemoglobin (blood)  BIOCHEMICAL FUNCTIONS OF PROTEINS Regulation – for regulation/coordination of bodily activities e.g. insulin (Beta cells pancreas), glucagon (Alpha cells pancreas) Receptor – for response of cell to external stimuli e.g. neuron receptors in nerve cells Contractile – for movement e.g. myosin, actin (muscles) Defensive – for protection against disease e.g. antibodies (blood)  BIOCHEMICAL FUNCTIONS OF PROTEINS RESERVOIR: Provides a reservoir of nitrogen and other nutrients, especially when external sources are low or absent o Ferritin, Ovalbumin, Casein AMINO ACIDS The building blocks for proteins Contains the following FG: H — Amino group (-NH2) H2N – C – COOH — Carboxyl group (-COOH) R — Side chain group (-R) General Structure of Amino Acids All the known amino acids are α-amino acids AMINO ACIDS All amino acids have at least one stereocenter (α-C) and are chiral (except Glycine). 2 Stereoisomers (enantiomers) o Laevus or L- o Dexter or D- Remember this? POP QUIZ! Given the amino acids, name the correct designation for the enantiomer. - - - Isoleucine Cysteine Tyrosine  AMINO ACIDS R side chain vary in size, shape, charge, acidity, functional groups present; and H-bonding ability and chemical reactivity Based on common “R” groups, there are 20 AAs 20 COMMON AMINO ACIDS Nomenclature Trivial names – assigned names Systematic names – amino- carboxylic acid 20 COMMON AMINO ACIDS Nomenclature Three-letter abbreviations (used for naming) — First letter of AA name is compulsory and capitalized followed by the next two letters not capitalized except for Asparagine (Asn), Glutamine (Gln) and Tryptophan (Trp) One-letter symbols (used for comparing AA sequences in proteins) — Usually the first letter of the name — When more than one AA has the same letter, the most abundant AA gets the first letter  20 COMMON AMINO ACIDS Abbreviations Abbreviations Amino Acid Amino Acid 3-letter 1-letter 3-letter 1-letter Alanine Ala A Leucine Leu L Arginine Arg R Lysine Lys K Asparagine Asn N Methionine Met M Aspartic acid Asp D Phenylalanine Phe F Cysteine Cys C Proline Pro P Glutamic acid Glu E Serine Ser S Glutamine Gln Q Threonine Thr T Glycine Gly G Tryptophan Trp W Histidine His H Tyrosine Tyr Y Isoleucine Ile I Valine Val V POP QUIZ A. Find the mystery word from the AA sequence of the peptides. 1. Trp-His-Ala-Thr 2. Phe-Ala-Met-Glu B. From the one-letter representation of the AA, give the name of the peptides. 1. VIA 2. CHEM THE 20 COMMON AMINO ACIDS Small Glycine, Alanine, and Serine Have few R groups since these are basic structures (contain alpha carbon, carboxylic acid, and amino group) Medium Valine, Proline, Asparagine, Cysteine, Threonine, and Aspartate Large Phenylalanine, Tyrosine, Histidine, Methionine, Leucine,Tryptophan, Glutamine, Isoleucine,Glutamate, Arginine, and Lysine  CLASSIFICATION OF AMINO ACIDS (based on R group) Non-polar (9) – hydrophobic, found in the interior of a protein. These are F, L, I, P, V, M, A, W, G Polar neutral (6)– uncharged, contain polar but neutral side chains. These are S, C, T, Q, Y, N Polar Acidic (2) – contain –COOH as part of the side chain. These are E, D Polar Basic (3)– contain –NH2 as part of the side chain. These are R, H, K NONPOLAR AMINO ACIDS POLAR NEUTRAL AMINO ACIDS POLAR ACIDIC AMINO ACIDS “Relatives” (Polar Neutral) POLAR BASIC AMINO ACIDS  OTHER CLASSES The ‘Aromatic’ WYF The ‘Sulfurated’ MC The ‘Lonesome’ G The ‘Branched’ VIL POP QUIZ! Classify the following AAs based on the polarity of the R-groups PA NP ALANINE PN TYROSINE Aspartic c. b. a. d. e. PB ARGININE PHENYLALAMINE Can you name them too?  CLASSIFICATION OF AMINO ACIDS (based on nutritional requirements) Essential (10) – a standard AA needed for protein synthesis, obtained from dietary sources because the body cannot synthesize it in adequate amounts from other substances - F, V, T, W, I, M, H, R, L, K - R is required in children but is not essential for adults. ESSENTIAL AMINO ACIDS Standard mnemonic: PVT TIM HALL F V T WIM H R L K (Exclusively) Ketogenic – can lead to ketone bodies – LL Both glucogenic and ketogenic – can lead to glucose or ketone bodies – PITTT (Exclusively) Glucogenic – can lead to glucose – all else CLASSIFICATION OF AMINO ACIDS (based on nutritional requirements) Nonessential (10) – a standard AA needed for protein synthesis but can be synthesized by the body. - A, R, N, D, C, E Q, G, P, S, Y - R is required in children but is not essential for adults. Amino Acid Sources Complete Dietary protein – contains all essential AAs in the amounts the body needs. Incomplete Dietary protein – does not contain adequate amounts of AAs the body’s needs. Limiting AAs is an essential amino acid that is missing, or present in inadequate amounts, in an incomplete dietary protein. Complementary Dietary protein – inc dietary protein + inc dietary protein = adequate amount of all essential AAs. DERIVED AMINO ACIDS Known as “nonstandard” amino acids Formed by enzyme-facilitated reaction on a common AA after that AA has been incorporated in a protein structure e.g. cysteine, desmosine and isodesmosine hydroxyproline and hydroxylysine found in collagen gamma-carboxylglutamate found in prothrombin BREAK! BREAK! BREAK! Have you heard of the following? Dopamine Norepinephrine These are neurotransmitters in psychiatric conditions, other Epinephrine pathologies and drug action and Histamine will be discussed in other Serotonin subjects. (closest one being Medicinal Chemistry) Melatonin BREAK! BREAK! BREAK! Catecholamines Dopamine Norepinephrine Tyrosine Epinephrine BREAK! BREAK! BREAK! Histidine BREAK! BREAK! BREAK! Serotonin Tryptophan Melatonin  PEPTIDES Are a chain of covalently linked amino acids. An unbranched chain of amino acids. Classified based on number of amino acids present - Dipeptide (2AAs), Tripeptide (3AAs), Oligopeptide (10 to 20 AAs), & Polypeptide (long chain of unbranched AA) AAs are joined together by peptide bonds.  PEPTIDE BONDS Remember how an amide is formed? In the same way peptide bonds are formed -Individual amino acids can be linked by forming covalent bonds between the –COOH and the –NH2 group. Water is eliminated. PEPTIDE BONDS Represented beginning with the AA with a free NH2 group (N- terminal end) and the other end contains free carboxyl group (C- terminal end) An amino acid residue is the portion of an amino acid structure that remains after the release of water when it dissociates from a peptide chain. BASIC PEPTIDE ANATOMY Glycine Alanine Serine This tripeptide has 2 peptide bonds. Name: Glycylalanylserine Draw the dipeptides glycylalanine and alanylglycine. Gylcylalanine (GA) Alanylglycine (GA) The two dipeptides are constitutional isomers, i.e. made up of the same AA but different order. Are they one and the same protein? NO! They are totally different in chemical and physical properties. Thus, the sequence of AA in peptides and peptides in proteins are very important.  Oxytocin and vasopressin are nonapeptides with 6 AA held in a loop by S-S (disulfide) bond from 2 Cysteine residues. Differs in position 3 an 8. Take note that the OH of the COOH at the terminal (Glycine residue) was replaced by –NH2  Enkephalins are pentapeptides. Two best known are Met- enkephalin and Leu-enkephalin. The two differ only in the last AA residue as their names imply. Met-enkephalin Leu-enkephalin Tyr-Gly-Gly-Phe-Met (YGGFM) Tyr-Gly-Gly-Phe-Leu (YGGFL)  Glutathione is a tripeptide. Glu (E) is bonded to Cys (C) through the –COOH side chain and not at the α-COO- Other perspective: Small peptide L-Aspartyl-L-phenylalanine methyl ester AMINO ACID INTERACTIONS Redox Oxidation: addition of O and removal of H Reduction: addition of H and removal of O ACID-BASE PROPERTIES OF AMINO ACIDS In pure form, AAs are white crystalline solids Most decompose before melting Not very soluble in water Under physiological conditions, exists as zwitterions The species with ZERO net charge is called zwitterion or double ion ACID-BASE PROPERTIES OF AMINO ACIDS AAs can exist in 3 forms (zwitterion & ionized - positive ion and negative ion) when in solution Equilibrium shifts with change in pH ACID-BASE PROPERTIES OF AMINO ACIDS Ionization (pH shifts) Condition Acidic Group (-COOH) Basic Group (-NH2) Process pH < pKa Neutral (COOH) Ionized to positive (NH3+) Protonated pH > pKa Ionized to negative (COO-) Neutral (NH2) Deprotonated ACID-BASE PROPERTIES OF AMINO ACIDS Out of the 20 common amino acids, 7 have ionizable groups ACID-BASE PROPERTIES OF AMINO ACIDS Isoelectric point (pI or ipH) – pH at which the concentration of zwitterion is at its maximum. - Different AAs have different ipH (refer to Stoker) - Nonpolar or polar neutral have ipH 4.8-6.3 - Polar basic – high ipH - Polar acidic – low ipH - At ipH, AAs are not attracted to applied electric fields because they carry net zero charge. ACID-BASE PROPERTIES OF AMINO ACIDS How do you determine ipH? What about those with R groups that are ionizable?  PEPTIDE ipH Steps: 1. Draw the peptide with the ionizable groups. 2. Protonate/deprotonate all ionizable groups. 3. Find the net charge (max charge) 4. Determine how many units away from zero is the max charge. 5. Find pKa before and after the zero. 6. Average the pKa between the no net charge.  ENZYMES Pharmaceutical Biochemistry Lecture OUTLINE OF DISCUSSION 1. Chemical nature/role of enzymes 1. Enzyme structure 2. Coenzymes, their reactions and their vitamin precursors 3. Classification and nomenclature 2. Models for the behavior of allosteric enzymes; Zymogens 3. Lock and Key; Induced Fit 4. Factors affecting enzyme activity 5. Reversible and Irreversible Inhibitors 6. Enzymes as markers for disease 7. Michaelis-Menten kinetic theory; Lineweaver-Burk Equation ENZYMES Biologic polymers that act as catalysts — increase the rate of reaction by lowering the activation energy (up to 1020 over uncatalyzed reactions) — are not used up or altered in the reaction Function under milder reaction conditions Efficient in catalyzing high reaction rate than chemical catalyst Except for ribozymes, majority of enzymes are proteins ENZYMES Structural classes Simple enzymes – purely protein (AA chain only) Conjugated enzymes – protein with non protein part Pepsin Cytochrome P450 Conjugated Enzyme An apoenzyme is the protein portion of the enzyme Most enzymes depend on use of cofactors The holoenzyme is the only active form of a conjugated enzyme ENZYME COFACTORS Inorganic metal ions are often found in Metal supplements as “trace Inorganic ions metals” or minerals Cofactor Most co-enzymes come Organic Coenzymes from water-soluble vitamins (particularly B vitamins) Cofactors may be permanently or temporarily bonded to the enzyme  ENZYMES and the METAL ION COFACTORS METAL ION ENZYME FUNCTION COFACTOR Cytochrome oxidase Cu +2 Redox Catalase Fe+2/Fe+3 Redox (H2O2) Alcohol dehydrogenase Zn +2 Used with NAD+ Carbonic anhydrase Zn +2 CO2 🡪 H2CO3 + HCO3- Carboxypeptidase A Zn +2 Hydrolyzes peptide bonds (COO) Glucose-6-phosphatase Mg +2 Hydrolyzes phosphate esters Arginase Mn +2 Removes electrons Urease Ni +2 Hydrolyzes amides General Characteristics of Vitamins Vitamin: An organic compound essential for proper functioning of the body Must be obtained from dietary sources because human body can’t synthesize them in enough amounts Needed in micro and milligram quantities – 1 gram of vitamin B is sufficient for 500,000 people Enough vitamin can be obtained from balanced diet Supplemental vitamins may be needed after illness Many enzymes contain vitamins as part of their structures - conjugated enzymes Two classes of vitamins – Water-Soluble and Fat-Soluble Synthetic and natural vitamins have the same function – 13 Known vitamins WATER-SOLUBLE VITAMINS VITAMIN COENZYME FORMED Vitamin C (ascorbic acid) n/a Vitamin B1 (thiamine) Thiamine pyrophosphate (TPP) Vitamin B2 (riboflavin) Flavin adenine dinucleotide (FAD+) Vitamin B3 (niacin/nicotinic acid) Nicotinamide adenine dinucleotide (NAD+) Vitamin B5 (pantothenic acid) Coenzyme A Vitamin B6 (pyridoxine) Pyridoxal phosphate (PLP) Vitamin B9 (folic acid) Tetrahydrofolate (THF) Vitamin B12 (cobalamin) Deoxyadenosylcobalamin (DAC) VITAMINS FAT-SOLUBLE Vitamin A (retin) – eyesight, skin Vitamin D (calciferols) – bone strength with the help of liver and kidney hydroxylases Vitamin E (tocopherols) – antioxidant Vitamin K (menaquinones) – coagulation WATER-SOLUBLE Vitamin C Vitamin Bs NOMENCLATURE OF ENZYMES Suffix –ase identifies a substance as an enzyme (some might still have –in e.g. trypsin, pepsin etc) Prefix is the type of reaction they catalyze e.g. oxidase, hydrolase Identity of the substrate is often noted in addition to the type of reaction. e.g. glucose oxidase, alcohol dehydrogenase Sometimes, the substrate is given rather than the reaction type. e.g. urease, lactase POP QUIZ! NOMENCLATURE OF ENZYMES Predict the action of the enzymes 1. Cellulase 1. Sucrase 1. L-amino oxidase 1. Aspartate aminotransferase 1. Lactate dehydrogenase CLASSIFICATION OF ENZYMES (IUB) EC #1 Oxidoreductases catalyze oxidations and reductions. EC #2 Transferases catalyze transfer of groups such as methyl or glycosyl groups from donor molecule to an acceptor molecule. EC #3 Hydrolases catalyze the hydrolytic (water-facilitated) cleaving of C – C, C – O, C – N, P – O, and certain other bonds, including acid anhydride bonds. EC #4 Lyases catalyze cleaving of C – C, C – O, C – N, and other bonds by elimination, leaving double bonds; or add groups to double bonds. EC #5 Isomerases catalyze geometric or structural changes within a single molecule. EC #6 Ligases catalyze the joining together of two molecules, coupled to the hydrolysis of a pyrophosphoryl group in ATP or similar nucleoside triphosphate. ENZYME CLASSIFICATION Main Class Selected Subclasses Type of Reaction Catalyzed EC#1 Oxidoreductases Oxidases Oxidation of a substrate Reductases Reduction of a substrate Dehydrogenases Introduction of double bond (oxidation) by formal removal of 2 H from substrate, the H is accepted by a coenzyme. EC#2 Transferases Transaminases Transfer of an amino group between substrates Kinases Transfer of a phosphate group between substrate ENZYME CLASSIFICATION Main Class Selected Subclasses Type of Reaction Catalyzed EC#3 Hydrolases Lipases Hydrolysis of ester linkage of lipids Proteases Hydrolysis of amide linkage in proteins Nucleases Hydrolysis of sugar-phosphate ester bonds in NA Carbohydrases Hydrolysis of glycosidic bonds in CHO Phosphatases Hydrolysis of phosphate-ester bonds EC#4 Lyases Dehydratases Removal of water from substrate Decarboxylases Removal of CO2 from substrate Deaminases Removal of NH3 from substrate Hydratases Addition of water to a substrate ENZYME CLASSIFICATION Main Class Selected Subclasses Type of Reaction Catalyzed EC#5 Isomerases Racemases Conversion of D to L isomer or vice versa Mutases Transfer of FG from one position to another in the same molecule EC#6 Ligases Synthetases Formation of new bond between two substrates, with participation of ATP Carboxylases Formation of new bond between a substrate and carbon dioxide, with participation of ATP EC#1 OXIDOREDUCTASE (Reductases, Oxidases, Dehydrogenases) Catalyze oxidation-reduction reactions, requires 2 substrates. EC # 2 TRANSFERASES (Transaminases, Kinases) Catalyze the transfer of a functional group from one molecule to another. + + EC#3 HYDROLASES (Lipases, Proteases, Nucleases, Carbohydrases, Phosphatases) Catalyzes a hydrolysis reaction in which the addition of a water molecule to a bond causes the bond to break EC#4 LYASES (Dehydratases/ Hydratases, Decarboxylases, Deaminases) Catalyzes the addition of a group to a double bond or the removal of a group to form a double bond in a manner that does not involve hydrolysis or oxidation EC#5 ISOMERASE (Racemases, Mutases, Epimerases) Catalyzes the isomerization (rearrangement of atoms) of a substrate  EC#6 LIGASE (Synthetases, Carboxylases) Catalyzes the bonding together of two molecules into one with the participation of ATP Enzymes are specific. Absolute specificity - catalyzes only one substrate. -e.g. catalase for H2O2 Stereochemical specificity - an enzyme can distinguish between stereoisomers. Chirality is inherent in an active site, because amino acids are chiral compounds. -e.g. L-amino acid oxidase Group specificity- structurally similar compounds that have the same functional groups. -e.g. carboxypeptidase for carboxyl end peptide linkages Linkage - involves a particular type of bond, irrespective of the structural features in the vicinity of the bond. -e.g. phosphatase for ester bonds of any phosphate esters ENZYME FUNCTION An enzyme may catalyze the conversion of one or more compounds (substrates) into one or more different compounds (products) and enhance the rates of the corresponding non catalyzed reaction by factors of at least 106 up to 1020 The enzyme and substrate must bind to an active site before any catalysis occurs  THEORIES OF ENZYME BINDING LOCK-AND-KEY MODEL The shape of the substrate and the conformation of the active site are complementary to one another. INDUCED-FIT MODEL The shape of the active site becomes complementary to the shape of the substrate only after the substrate binds to the enzymes. THEORIES OF ENZYME BINDING ENZYME ACTIVITY A measure of the rate at which an enzyme converts a substrate into a product in a biochemical reaction. It is affected by: -Temperature -pH -Substrate concentration -Enzyme concentration FACTORS AFFECTING ENZYME ACTIVITY Temperature Measure of kinetic energy (KE) of molecules At ↓T: ↓KE, less molecular collisions, ↓reaction rate ↑T beyond optimum denatures the enzyme Optimum temperature is the temperature at which an enzyme exhibits maximum activity FACTORS AFFECTING ENZYME ACTIVITY pH Measure of acidity of a system Slight change in pH alters the charge in the acidic and basic amino acid residues Extreme pH denatures the enzyme Optimum pH is the pH at which an enzyme exhibits maximum activity  FACTORS AFFECTING ENZYME ACTIVITY Substrate concentration Enzyme activity increases only to a certain substrate concentration and there after remains constant Turnover number is the number of substrate molecules transformed per minute by one molecule of enzyme under optimum conditions of T, pH and saturation. FACTORS AFFECTING ENZYME ACTIVITY Enzyme Concentration If the amount of enzyme is increased, the reaction rate also increases. FACTORS AFFECTING ENZYME KINETICS What is the normal body temperature? Blood pH?  ENZYME MODULATION There are molecules (not cofactors) that affect enzyme activity. May target an active site or another cavity somewhere in the enzyme called an allosteric site — Activator (uncommon for drugs) — Inhibitor  ENZYME INHIBITION Reversible Competitive Inhibition Occurs when a molecule (inhibitor) that sufficiently resembles the S in shape and charge that it can compete with the S for the active site. Effect: blocks the reaction, slows it down. Inhibition: Reversible  ENZYME INHIBITION Reversible Noncompetitive Inhibition Occurs when a molecule (inhibitor) that decreases enzyme activity bind to a site on an enzyme other than the active site. Effect: decrease enzyme activity, slows it down. Inhibition: Reversible  ENZYME INHIBITION Reversible Uncompetitive Inhibition Occurs when a molecule (inhibitor) binds to the E/S complex and prevents the conversion of the substrate into a product. Effect: decrease enzyme activity, slows it down. Inhibition: Reversible  ENZYME INHIBITION Irreversible Inhibition Occurs when a molecule inactivates the enzyme by forming strong covalent bond to an amino acid R group at the enzymes active site. Effect: blocks the reaction Inhibition: Irreversible REGULATION OF ENZYME ACTIVITY Enzymes need to be “turned off” for the cell to conserve energy. Mechanisms Feedback control Proteolytic enzymes and zymogens Covalent Modification REGULATION OF ENZYME ACTIVITY Most enzymes responsible for regulating cellular processes are allosteric enzyme. — Has quaternary structure — Has 2 kinds of binding sites: 1) substrate 2) regulators — Binding at the regulatory site changes the 3D structure of the enzyme and change the shape of the active site. ▪ Increase ▪ Decrease Effects of positive and negative regulators REGULATION OF ENZYME ACTIVITY Mechanisms of regulation 1. Feedback control 2. Timed “on-off” 3. Covalent modification of enzyme MECHANISM OF ENZYME REGULATION 1. Feedback Control A process in which activation or inhibition of the first reaction in a sequence of reactions is controlled by a product of the reaction sequence. MECHANISM OF ENZYME REGULATION 2. Proteolytic enzyme and zymogens (Timed “on-off”) Production of inactive form of proteolytic enzymes (zymogens or proenzyme) which are activated (turned “on”) at the appropriate time. MECHANISM OF ENZYME REGULATION 3. Covalent Modification Process where enzyme activity is altered by covalently modifying the structure of the enzyme through attachment of a chemical group to or removal of a chemical group from a particular amino acid within the enzyme’s structure. - Phosphorylation and dephosphorylation of some enzymes DRUGS AS ENZYME INHIBITORS Sulfonamides – inhibit use of p-aminobenzoic acid (PABA) in bacterial synthesis of folic acid -> antibiotic action Penicillins – inhibit transpeptidase enzymes for bacterial cell wall synthesis -> antibiotic action Quinolones (floxacins) – inhibit topoisomerase/gyrase enzymes needed for proper DNA synthesis -> antibiotic action Cholinesterase inhibitors – inhibit acetylcholinesterase to improve cholinergic activity Food-Enzyme-Drug Interactions Liver enzymes cytochrome P450 are involved in drug metabolism. There are foods and drugs that inhibit or induce the activity of an enzyme. — “Grapefruit effect” slows down metabolism of some drugs — Rifampicin and phenytoin speeds up metabolism of verapamil and diltiazem. ENZYMES AS MARKERS OF DISEASE REVIEW OF CHEMICAL KINETICS Factors affecting chemical reactions: Concentration of reactants Temperature V Surface area Nature of reactants Presence of catalysts [S]  ENZYME KINETICS Assumptions: The more enzyme, the more substrates [S] acted upon The more substrates converted to product, the faster the enzyme activity (“enzyme velocity” or [V]) A direct proportion (straight line x vs. y) ENZYME KINETICS General idea: Our enzymes are LIMITED. Our enzymes are SATURABLE. E + S ↔ E/S → E + P The curve is hyperbolic. Linear at low S Independent of S at high S BUT WHY DOES [S] vs [V] look like this? The MICHAELIS-MENTEN GRAPH Vmax – represents highest attainable velocity Km (Michaelis constant) – reflects concentration of substrate needed to reach half of Vmax — Reflective of how good the enzyme and substrate go together (“affinity”) — Lower Km means less concentration needed to get the same result – better affinity THE MICHAELIS-MENTEN GRAPH Problem of using the Michaelis-Menten graph: Hard to accurately determine Vmax (due to the curve) Solution: Use other types of graphing that deal with using lines Examples: Lineweaver-Burk plot Eadie-Hofstee plot Hanes-Woolf plot THE LINEWEAVER-BURK PLOT Uses the reciprocal of both [S] and [V], thus it is also called the double reciprocal plot Basic math: Magnitude inverts position when reciprocals are plotted Higher from point 0 = smaller value, etc. Easier way to spot Vmax and Km COMPARISON  Vmax Km Competitive Same Increases Noncompetitive Decreases Same Uncompetitive Decreases Decreases  Vmax Km Competitive Same Increases Noncompetitive Decreases Same Uncompetitive Decreases Decreases SUMMARY Enzymes are biological catalysts Enzymes are specific and saturable Enzymes are either simple or conjugated with cofactors Enzymes are classified by the reaction they catalyze Enzyme kinetics can be shown using many types of plots (ex. MM, LB) Temperature and pH can affect enzyme activity Enzyme activity is modulated by activators or inhibitors. Inhibitors may work competitively, noncompetitively, or uncompetitively Enzyme modulation may also be due to covalent modification or negative feedback Enzyme inhibitors are often seen in drugs Enzymes may be used as aid for clinical diagnosis of disease