Enzymology-P1- Intro-History-Structure PDF

Faculty of Women for Arts, Science & Education Ain Shams University Botany Department Microbiology & Chemistry Enzymology Biophysics Programs BOT 332 (Credit Hours)...

Faculty of Women for Arts, Science & Education Ain Shams University Botany Department Microbiology & Chemistry Enzymology Biophysics Programs BOT 332 (Credit Hours) Third level Prepared by : Fifth semester Dr Abeer A. Rushdy Professor of Microbiology Dr Walaa Nabel Lecture of Microbiology  Contents Part I Part V 1) Introduction How do enzymes work? 1) Mechanism of enzyme-catalyzed reaction 2) Etymology & History 2) Enzymes have an Active Site 3) Importance of enzymes 3) Substrate specificity 4) Location of enzymes 4) Factors Affecting Enzyme Activity Part II Part VI 5) Enzyme Function / Enzymatic Catalysis / 1) Enzyme Structure Catalytic Activity Part III - Free energy - Activation energy - 1) Enzyme Nomenclature Transition state 2) Classification of Enzymes 6) Enzyme Kinetics 7) Enzyme Regulation Part IV Part VII 1) General properties of the enzyme 1) Isolation and Purification of Enzymes  Enzymes are very important for the sustainability of life in all life forms. Enzymes are key to life History of Enzymes Etymology & History Enzymology had a glorious past, both in terms of recognition of the studies (several Nobel prizes were awarded) as well as commercialization (a large enzymology industry exists). Marketing References from the writings of various ancient civilizations from Babylon, Rome, Greece, Egypt, China, and India suggest the use of microorganisms as enzyme sources for fermentation was widespread among ancient people. fermentation Description of wine making in the oldest known reference to the commercial use of enzymes in the Codex of Hammurabi from ancient Babylon civilization around, 2100 BC. Process of vinegar production, which is based on the enzymatic conversion of alcohol to acetic acid. Vinegar is used for food storage and preparation and medicinal purposes. Dairy products were an important food source in ancient societies. The manufacture of, bread, and alcoholic beverages, in the processes known as fermentation Book-Understanding Enzymes- An Introductory Text-2018- P19 History of Enzymes Etymology & History Discovery of first enzymes, By the late 17th and early 18th centuries, - The digestion of meat by stomach secretions - The conversion of starch to sugars by plant extracts and saliva However, the mechanism by which this occurred had not been identified. Anselme Payen (French chemist) - The first to discover an enzyme, diastase, in 1833 (converting starch into sugar) - It was extracted from malt solution - Today, diastase refers to ,  or  -amylase that can break down carbohydrates https://www.slideserve.com/pete/enzyme-an-essential-catalyst-there-are-two-fundamental-conditions-for-life-powerpoint-ppt-presentation https://www.slideshare.net/slideshow/enzimas-industria/250508458 https://cellltheory.weebly.com/theodor-schwann.html History of Enzymes Etymology & History Theodor Schwann In 1834, The German biologist - Considered a founder of the cell theory and experimented to disprove spontaneous generation. - He discovered pepsin, a gastric juice that can digest food in a test tube (the first digestive enzyme prepared from animal tissue). Jöns Jacob Berzelius In 1835, Swedish chemist - In 1835 Jöns Jakob Berzelius recognized that some substances in living bodies cause chemical reactions but do not themselves undergo change (catalytic nature). He named them catalysts from the Greek word katalyein, (Greek : to dissolve). - It was suspected that biological catalysts are involved in the fermentation of sugar to form alcohol (hence the name "ferments"). https://www.slideserve.com/pete/enzyme-an-essential-catalyst-there-are-two-fundamental- conditions-for-life-powerpoint-ppt-presentation https://www.slideshare.net/slideshow/enzimas-industria/250508458 History of Enzymes Etymology & History Louis Pasteur , In 1850, a French chemist, pharmacist, and microbiologist - While studying the fermentation of sugar to alcohol by yeast, He concluded that this process was driven by a "vital force" within yeast cells (function only within living organisms), called "ferments". i.e. The fermentation process is inseparable from living cell Wilhelm Kühne, In 1876, German physiologist - Studying catalysis in yeast extracts - First used the term enzyme - The word ‘enzyme’ was first coined in 1876 by the Wilhelm Kühne, from Ancient Greek ἐν (en,"in") + ζ´ νµε (zúm¯e, = Greek word enzymos which means "leavened" or "in yeast"). - It refers to the substances used by alive yeast cells to carry out the fermentation of sugars to alcohol. - This reflects the early belief that these catalytic agents were tied to yeast's biological This reflects the early belief that these catalytic agents were tied to yeast's biological activity. https://www.slideserve.com/pete/enzyme-an-essential-catalyst-there-are-two-fundamental-conditions-for-life-powerpoint-ppt-presentation activity. https://www.slideshare.net/slideshow/enzimas-industria/250508458 https://cellltheory.weebly.com/theodor-schwann.html History of Enzymes Etymology & History Eduard Buchner, In 1896, German biochemist - Discovers that cell-free yeast extracts can ferment sugar (lacking any living yeast cells), proving that enzymes can function outside living cells. i.e. Cell-free extract, prepared from yeasts by filtration, contained active enzyme - He named the enzyme that brought about the fermentation of sucrose "zymase“ (extracted from yeast cells). Awarded Nobel Prize for Chemistry (1907) Victor Henri, in 1903, a French-Russian physical chemist - Concluded that an enzyme combines with its substrate to form an enzyme- substrate complex as an essential step in enzyme catalysis. https://www.slideserve.com/pete/enzyme-an-essential-catalyst-there-are-two-fundamental-conditions-for-life-powerpoint-ppt-presentation https://www.slideshare.net/slideshow/enzimas-industria/250508458 https://cellltheory.weebly.com/theodor-schwann.html History of Enzymes Etymology & History Michaelis and Menten, a German man and a Canadian woman, in 1912. - The general theory of enzyme action was expressed mathematically by Leonor Michaelis and Maud Leonora Menten - The Michaelis–Menten equation is mainly used to characterize the enzymatic rate at different substrate concentrations They postulated that the enzyme (E) first combines with its substrate (S) to form an enzyme-substrate complex (ES) in a relatively fast reversible reaction: E + S =ES The ES complex then breaks down in a slower reversible reaction to yield the reaction product (P) and the free enzyme (E): ES = P + E https://www.slideserve.com/pete/enzyme-an-essential-catalyst-there-are-two-fundamental-conditions-for-life-powerpoint-ppt-presentation https://www.sciencehistory.org/education/scientific-biographies/leonor-michaelis-and-maud-leonora-menten/ History of Enzymes Etymology & History James Sumner 1926 - First obtained crystals of an enzyme (in pure form), urease from jack bean, and proved it to be a protein protein. (Nobel Prize in Chemistry in 1946) John Northrop 1929 - Isolation and crystallization of trypsin and pepsin pepsin were done - Who also proved the enzymes pepsin and trypsin are proteins (Share in the Nobel Prize in chemistry in 1946) https://onlinelibrary.wiley.com/doi/abs/10.1002/9781119793304.ch1 https://www.slideshare.net/slideshow/history-of-enzymes/82121542 https://www.sciencephoto.com/media/228536/view/james-b-sumner-american-biochemist History of Enzymes Etymology & History In the 18th and 19th centuries, scientists started systematically studying enzyme actions. Today, enzymes are vital in various food and beverage manufacturing processes and consumer products. Enzymes hold significance in Health Sciences, as several disease processes are associated with abnormal enzyme activities and in Clinical Enzymology (Diagnosis, and Therapeutics). The study of enzymes remains crucial to science and society.  Importance of enzymes Enzymes exist in all biological systems in abundant numbers, enabling many vital biochemical reactions to proceed at a speed compatible with life.  Enzymes speed up chemical reactions within each cell.  Enzymes facilitate various biochemical reactions (Metabolism) necessary for their survival, and growth.  Enzymes are essential for physiological functions such as respiration, fermentation, digestion, reproduction, muscle and nerve function, and more. Too much or too little of a certain enzyme can cause problems (health) Reactions in cells are different from general chemical reactions: easily, rapidly, regulated, and controlled Enzymes play an important role in Clinical enzymology (Diagnosis, and Therapeutics) Enzymes enable cells of microorganisms to ecolgicl success and adapt to various environmental conditions In pathogenic organisms, enzymes (such as proteases, lipases,...) contribute to their ability to infect hosts by evading the immune system, and by antibiotic resistance, thereby allowing bacteria to survive in the presence of these drugs. The enormous diversity of microbe enzymes makes them a motivating cluster of products for application in several areas like agriculture, industry, food processing, textile industry, pharmaceuticals, wood processing, cosmetics, and environmental pollution control as bioremediation and biodegradation.  Location of enzymes Enzymes are found in all living cells, all tissues, and fluids of the body In unicellular, and multicellular In unicellular organisms, organisms & multicellular prokaryotes & eukaryotes Enzymes in cells can be located in various cellular compartments or specific organelles within the cell. Enzymes are not always found uniformly within a cell; may formed only when required. The rates of enzyme synthesis are influenced by different factors (Gene Regulation, Nutrient Availability, Environmental Factors, Hormonal Regulation, Stress Responses,…) Enzymes can be intracellular intracellularand or extracellular extracellular. Enzyme location controls enzyme activity  Location of enzymes Intracellular enzymes (Endoenzymes) ▪ Produced in the cell, and are always active, performing functions within the cell. ▪ Substrate, Products, and active enzymes, are inside the cell ▪ Functions to further break down nutrients that come into the cell to yield energy for driving cellular functions OR synthesis of cellular components by forming building blocks ▪ The majority of enzymes fall within this category Extracellular enzymes (Exoenzymes) ▪ Produced in the cell in an inactive form and sent outside (become active and function outside the cell). ▪ Substrate, Products, and active enzymes are outside the cell ▪ Participates in the breakdown of larger macromolecules https://slideplayer.com/slide/7609951/ Overview Part II Enzyme Structure Enzyme Structure ii. Non-Proteinaceous enzyme i. Proteinaceous enzyme ▪ Ribozymes 1. Simple-protein enzymes Classification of enzymes according 2. Conjugated enzyme to the polypeptide chain A protein part ▪ Monomeric enzymes A nonprotein part (Cofactors) ▪ Oligomeric Enzymes ▪ Inorganic ions (Essential ions) Multienzyme Complexes - Metalloenzymes Metallozymes Multifunctional Enzymes - Metal-activated enzymes Isoenzymes - Isozymes ▪ Organic molecules (Coenzyme) - Co-substrates - Prosthetic groups.  Enzyme Structure Chemically, enzymes are protein catalysts that catalyze chemical reactions in biological systems With the exception of catalytic DNA and RNA (Ribozymes) which are considered enzymes but not proteins ✓ Enzymes are the largest class of proteins. Since the large majority of enzymes are proteins, ✓ Enzymes are found as globular proteins, which we’ll start by looking at protein structure. form a spherical shape. Polypeptide chain ✓ Enzymes are large molecules, the molecular weights of which range from several thousand to several million ✓ Enzymes made of long chains of amino acids polypeptide chain called polypeptide chain (from 62 to an average of 2500 amino acid residues) that fold over each other (folding to form a tertiary structure held together by a range of bond types between R- groups or ‘side- chains’) giving rise to a globular structure with a unique three-dimensional structure https://en.wikiversity.org/wiki/Enzyme_structure_and_function  Enzyme Structure Chemically, enzymes are protein catalysts that catalyze chemical reactions in biological systems With the exception of catalytic DNA and RNA (Ribozymes) which are considered enzymes but not proteins Polypeptides consist of amino acids joined by a Since the large majority of enzymes are proteins, peptide bond in the same chain we’ll start by looking at protein structure. 3D due to certain amino acids with SH groups forming disulfide (S-S) bonds with other amino acids may in the same chain or other interactions between R groups of amino acids are non-covalent interactions such as hydrogen bonds, and ionic bonds,… ✓ The unique configuration of each enzyme (select from 20 amino acids combined in various sequences) enables it to find the correct substrate from among the large number of diverse molecules in the cell, giving the specificity, which in turn identifies the catalytic activity of the enzyme. All enzymes are proteins, but all proteins are not enzymes Produced by ribosomal RNA  Enzyme Structure Proteinaceous enzyme Chemically, the enzymes may be divided into 2 categories: Complex enzymes Simple enzymes Conjugated enzyme ▪ They are only made up of ▪ They consist of a protein part and a proteins non-protein part (cofactors) essential for the activity https://www.quora.com/How-are-conjugated-enzymes-defined https://www.researchgate.net/publication/346517277_Enzymatic_Glucose-Based_Bio-batteries_Bioenergy_to_Fuel_Next-Generation_Devices/figures?lo=1 https://pdb101.rcsb.org/motm/74  Enzyme Structure Simple enzymes Many enzymes are simple proteins (called apoenzymes) that are formed from one or more chains of polypeptides bound together to form a large molecule that folds into a three- dimensional shape or tertiary structure without any non-protein components ▪ Apoenzymes alone are active enzymes. ▪ Such as Urease, Amylase, digestive enzymes (trypsin, pepsin, chymotrypsin), etc. Urease Amylase Pepsin Chymotrypsin https://en.wikipedia.org/wiki/Amylase https://www.researchgate.net/publication/349501668_A_Review_of_Enzyme_Induced_Carbonate_Precipitation_EICP_The_Role_of_Enzyme_Kinetics/figures?lo=1  Enzyme Structure Complex enzymes Conjugated enzyme ▪ When enzymes have both protein (amino acids) and non-protein components. ▪ Such as Catalase, Cytochrome c, lipoproteins, glycoproteins, etc. A protein part A nonprotein part A protein part called Apoenzymes are Some enzymes require an important for enzymatic activity since additional nonprotein component they are responsible for the specificity called the cofactor. of enzymes to their substrates. The cofactor can be of two Apoenzymes alone are not active types:- enzymes; they must bind to cofactor to - Inorganic ions (metal ions) be activated. - Organic group  Enzyme Structure Complex enzymes Conjugated enzyme The Conjugated enzyme without its non-protein moiety is termed an apoenzyme and is inactive. Enzyme protein alone = Apoenzyme Inactive Apoenzyme + Cofactor = Holoenzyme Active Holoenzyme consists of an apoenzyme together with its cofactors (non-protein component). Holoenzyme is a complete, functional enzyme (active), which is catalytically active. Enzyme Structure A nonprotein part (Cofactors) ✓ Cofactors are non-protein substances that are associated with enzymes (mostly found at the active site). ✓ Don’t interact with the enzymes or substrates ✓ There are two types of cofactors: Inorganic ions (Essential ions – Metal ions) like Fe2+, Zn2+, Mg2+ etc. Organic molecules (Coenzyme) is a small organic molecule. ✓ The key activities of enzyme cofactors (functions) 1. Stabilizing the structure (3D) of the enzyme 2. Essential for the functioning of some enzymes and to show their full activity ✓ Create favorable conditions for reactions to occur by lowering activation energy, participating in redox reactions, helping in electron transport, etc. ✓ Assist the substrate in binding and interaction with the enzyme which orients the substrate properly for reaction, Transfer of functional groups, etc. https://www.biologybrain.com/examples-of-cofactors-and-coenzymes/cofactors-examples/#main Enzyme Structure Family tree of cofactors A nonprotein part (Cofactors) Metalloenzymes Metallozymes Inorganic ions (Essential ions) Metal-activated enzymes Cofactors (A nonprotein part) Co-substrates Organic molecules (Coenzyme) Prosthetic groups. Enzyme Structure Metal cofactors A nonprotein part Inorganic ions (Cofactors) Essential ions The two major categories 25-30% of all enzymes 1. Ions in Metalloenzymes or Metallozymes require a metal ion as a cofactor 2. Activator ions in Metal-activated enzymes The cofactors are attached to the apoenzyme (tightly or loosely bound) and are not consumed in the reaction. Enzyme Structure Metal cofactors A nonprotein part Inorganic ions (Cofactors) Essential ions 1. Ions in Metalloenzymes or Metallozymes ✓ Metalloenzymes are enzymes that require metal ions (usually divalent or more) for their structural stability, and catalytic function. ✓ The metal ions are firmly bound (Enz-M) in the active site and cannot be removed without disrupting the apoenzyme architecture (denaturation), as it forms covalent bonds with the amino acids of the enzyme. ✓ Sometimes, enzymes require more than one metal ion for their activity (same or different) ✓ Adding more metal ions will not improve the catalytic efficiency https://pubs.rsc.org/en/content/articlehtml/2019/ob/c9ob01091b Enzyme Structure Metal cofactors A nonprotein part Inorganic ions (Cofactors) Essential ions 2. Activator ions in Metal-activated enzymes ✓ Metal-activated enzymes are enzymes that need metal ions (usually monovalent or divalent, as Na+, K+, Ca2+ and Mg2+) just for activation function (increased activity) that do not require metal ions for their structural integrity ✓ Activator ions are associated relatively weakly & loosely bound (Enz …M) to the enzyme and hence, can be removed without causing any denaturation or change in the 3D structure of the enzyme and these ions will not take part in the enzyme active site formation. ✓ In such enzymes, the metal ion requires only at the time of reaction to activate the enzymes or to complex with the substrate. ✓ These enzymes require metal ions in excess (around 2-10 times higher than the concentration of the enzyme) because they cannot bond with the metal ion permanently. ▪ Removing the metal from the vicinity of the enzyme causes it to lose activity, and adding back the metal ion is necessary to restore activity. ▪ The addition of more metal ions into the medium will positively affect the catalytic efficacy of the enzyme. Enzyme Structure Metal cofactors A nonprotein part Inorganic ions (Cofactors) Essential ions Examples of ions in metalloenzymes: Zn2+ ------------→ in carbonic anhydrase Structure of Cu2+, Zn2+, Mg2+ --------→ in cytochrome oxidase human cytochrome c Fe2+ , Fe3+ --------→ in catalase the enzyme binds metals such as Cu, Mg and Zn Examples of Metal-activated enzymes: Mg+2 or Mn2+ -----------→ Phosphotransferases Ca+2 ------------→ Thrombokinase K+ in pyruvate kinas K+ ------------→ Pyruvate kinase https://www.vedantu.com/question-answer/which-of-the-following-is-the-cofactor-of-class-12-biology-cbse-5f7447d40fd0025e776689bc https://www.researchgate.net/publication/266890071_Direct_Electron_Transfer_of_Cytochrome_c_on_ZrO_2_Nanoparticles_Modified_Glassy_Carbon_Electrode/figures?lo=1 https://www.researchgate.net/publication/357815483_The_Role_of_COA6_in_the_Mitochondrial_Copper_Delivery_Pathway_to_Cytochrome_c_Oxidase/figures?lo=1 Enzyme Structure Organic cofactors A nonprotein part Organic molecules (Cofactors) Coenzyme There are two classes of coenzymes 1. Co-substrates 2. Prosthetic groups. The coenzyme is smaller, organic molecules, low molecular weight, non-protein parts attached to the protein part of the enzyme Coenzymes can be either loosely or tightly bound to the enzyme. Coenzymes cannot themselves catalyze a reaction, but they can help enzymes to do so Coenzymes form complexes with enzymes (enzyme-coenzyme complex) In some enzymes, coenzymes are released from the enzyme during the chemical reaction which is altered during chemical reactions and is considered to be a type of secondary substrate https://www.learnpick.in/prime/documents/ppt/1414/enzyme Enzyme Structure Organic cofactors A nonprotein part Organic molecules (Cofactors) Coenzyme Most coenzymes are vitamins, vitamin derivatives, or Nucleotides, nucleotide derivatives ▪ A coenzyme is usually a form of activated vitamin that is essential for biochemical pathways such as catabolism, anabolism, and the production of energy and some of them are important as growth factors. ▪ All the water-soluble vitamins can act as coenzymes or coenzyme precursors which include Vitamins-B complex and Vitamin C (may vitamins A & K as fat- soluble that act as coenzymes). Most vitamins must be enzymatically transformed into the coenzyme A deficit of vitamins and as a result correspondent coenzyme results in the disease https://www.slideshare.net/slideshow/229983-lecture-26-114696566/114696566#7 Enzyme Structure Organic cofactors A nonprotein part Organic molecules (Cofactors) Coenzyme The function of coenzymes Prepares the active site for catalytic activity ▪ Providing additional reactive functional groups bind active sites, aid in enhancing catalytic activity. ▪ Bind to the allosteric site causing a conformational change that enables the active site to be functional for the substrate to bind. Assist the enzymes in biochemical transformations. Transfer of a specific group (functional groups), electrons (as electron carriers) between enzymes & substrates or from one substrate to another A coenzyme transports a variety of chemical groups ( such as Hydride, Acetyl, Formyl, Methyl) Hydride ions which are carried by coenzymes such as NAD, Phosphate groups which are carried by coenzymes such as ATP Acetyl groups which are carried by coenzymes such as coenzyme A. Enzyme Structure Organic cofactors A nonprotein part Organic molecules (Cofactors) Coenzyme How does a coenzyme work? -In coenzyme-assisted reactions The enzyme remains unchanged. The enzyme begins with a shape that is not fully complementary to the substrate. The structure of the coenzyme is changed. During the reaction, the coenzyme binds to the active site, donates energy or molecules, and cannot be immediately reused. The coenzyme binding ensures that the enzyme's shape becomes complementary. After the reaction, the coenzyme can be recycled by accepting energy (e.g. the bonds formed with electrons, H+, phosphate ions), so it can go on to assist in more reactions. Enzyme Structure Examples of Co-substrates Organic cofactors A nonprotein part Organic molecules (Cofactors) Adenosine triphosphate (ATP) Coenzyme Nicotinamide adenine dinucleotide (NAD) Coenzyme-A (Co-A) 1. Co-substrates Cosubstrates are coenzymes loosely bound to the enzymes and form the active site and can be removed from the enzyme without damaging the structure of the enzyme. Coenzyme is called a co-substrate because it binds to the enzyme along with the substrate at the beginning of a chemical reaction and is altered during the reaction (as part of the catalytic cycle), dissociates from the enzyme (apoenzyme) in an altered state, and are regenerated by other enzymes found in the cell to their original form to be reused Repeated recycling within cell Enter reaction, get altered, leave, regenerate, reused Enzyme Structure Examples of prosthetic group Organic cofactors A nonprotein part Organic molecules (Cofactors) Flavin adenine dinucleotide (FAD) Coenzyme Biotin 2. A prosthetic group Some enzymes contain a ‘built-in’ cofactor called prosthetic groups A prosthetic group is tightly bound to the enzyme, covalently and permanently bound, and remains bound during the reaction, eventually gets restored to the starting state (original form) and involved in active site formation The prosthetic group cannot be removed from the enzyme until the structure of the enzyme gets denatured. The prosthetic groups may be organic compounds such as a vitamin, sugar, or lipid. https://www.google.com/imgres?q=biotin%20prosthetic%20group& imgurl=https%3A%2F%2Fcdn.lecturio.com%2Fassets%2FProsthetic- group.png&imgrefurl=https%3A%2F%2Fwww.lecturio.com%2Fcon cepts%2Fbasics-of-enzymes%2F&docid=Jo1yyeQIglEzHM&tbnid=t- p8C6quXp  Enzymes Conjugate/complex Simple (Haloenzyme) Protein Non-protein (Apoenzyme) Cofactors Inorganic Organic (essential ions) (coenzymes) Tightly bound Metalloenzymes Co- Loosely bound or Metallozymes substrates Metal-activated Prosthetic Loosely bound enzymes Tightly bound groups  Enzyme Structure The macromolecular components of all enzymes consist of protein, except in the class of RNA catalysts called ribozymes. Non-proteinaceous enzyme Ribozymes The first ribozyme was discovered in 1980. A ribozyme (ribonucleic acid enzyme or RNA enzyme or catalytic RNA ) is an RNA molecule that can perform specific biochemical reactions, like enzyme activity in the absence of protein It contains an active site that consists entirely of RNA Researchers demonstrate that RNA functions both as a genetic material or as a biological catalyst. Ribozymes are capable of cleaving mRNA molecules in a sequence specific, catalytic manner. A small number of ribozymes exist which serve as an RNA-based biological catalyst. Example: Ribonuclease p (RNAse p) was the first true RNA enzyme identified mRNA translation, Peptidyl transferase https://www.slideshare.net/slideshow/ribozyme-technology/151347482#4 https://en.wikipedia.org/wiki/Ribozyme https://www.slideshare.net/VipinKannan1/ribozymes-55681679#3  Enzyme Structure Classification of enzymes according to the polypeptide chain Monomeric enzymes ▪ Enzyme is made up of one polypeptide chain in tertiary structure ▪ Most act without a cofactor As Simple proteins ▪ They are often synthesized in inactive form: proenzyme or zymogen ▪ Very few monomeric enzymes are known ▪ E.g. Proteases as trypsin, chymotrypsin, and pepsin…. Oligomeric Enzymes ▪ Two or more polypeptide chains linked usually by non-covalent interactions and NEVER by a peptide bond Dimeric proteins consist of two Trimeric proteins of three. Tetrameric proteins of four sub-units. ▪ Each polypeptide chain of such enzymes is called subunit or monomer unit ▪ May be identical or different subunits ▪ Not synthesized as inactive forma as zymogens ▪ The vast majority of known enzymes are oligomeric ▪ E.g lactate dehydrogenase (LDH), Lactose synthase, Hexokinase chrome-  Enzyme Structure Multienzyme Complexes and Multifunctional Enzymes ✓ Multienzyme complex Multienzyme system ▪ In a number of metabolic pathways, the bound of several enzymes together by non-covalent forces (form Metabolite tunneling) which may easily undergo dissociation and re- association. Multienzyme complex ▪ Catalyze a series of sequential biochemical reactions in the same pathway ▪ Can greatly increase the rate of reactions Examples: Pyruvate Dehydrogenase Complex; Electron transport Chain. Metabolite (Substrate) channeling or tunneling ✓ “Channeling” of reactants between active sites ✓ The product (intermediate) of one reaction is transferred directly to the next active site without entering the bulk solvent (do not diffuse away from the complex) ✓ Can greatly increase the rate of reactions chrome-  Enzyme Structure Multifunctional Enzymes The fatty acid synthase ▪ Enzymes contain multiple active sites (two or more) that complex has 7 active sites can use different substrates and perform different catalytic functions. ▪ May be form Metabolite tunneling ▪ Catalyze a series of sequential biochemical reactions of a metabolic pathway, product of one site is the substrate for the next site. ▪ Play a crucial role in metabolic pathways by increasing efficiency, as they can carry out several steps of a metabolic pathway without releasing the intermediate products into the surrounding environment. ▪ The presence of multiple activities on a single polypeptide Examples: The fatty acid synthase complex has 7 active sites chain is usually the result of a gene fusion event https://www.slideserve.com/xavier-potts/biochemistry https://www.youtube.com/watch?v=m5LGlvDVNvY https://biotechnologymcq.com/multifunctional-enzyme-fatty-acid-synthase/  Enzyme Structure Isozymes - Isoenzymes In the same organism, there are multiple forms of the same enzyme, isomers of the enzyme, which differ in amino acid sequence, catalyze the same chemical reaction in different tissues of the body. Oligomeric enzyme (i.e. forms from more than one type of subunits) In general, synthesized from different genes (distinct genetic sources) Different forms may be differentiated from each other based on certain distinct properties. ▪ Electrophoretic mobility ▪ Absorption properties ▪ Reaction with a specific antibody ▪ Kinetic properties (Km values) ▪ Regulatory mechanisms, ▪ Tissue distribution https://www.slideshare.net/slideshow/isoenzymes-59711638/59711638 https://www.biologybrain.com/isoenzymes/  Enzyme Structure Isozymes - Isoenzymes Example: Enzyme Lactate dehydrogenase LDH (five isoenzymes) E.C – 1.1.1.27 (oxidoreductase group) - LDH consists of four polypeptide subunits or monomers, thus called is a “tetrameric enzyme” - Tetrameric enzyme composed of two subunits, M-form, and H-form, resulting in five isozymes ▪ Two homotetramers: LDH-1 (4H), LDH-5 (4M), ▪ Three hybrid forms (mixed tetramers): LDH-2 (3H1M), LDH-3 (2H2M), LDH-4 (1H3M). - These five isoforms are enzymatically similar but show different tissue distributions (in the brain, lunge, kidneys, ……) Lactate dehydrogenase (LDH) E.C – 1.1.1.27 LDH exist in five varieties in Human body LDH-1 LDH-2 LDH-3 LDH-4 LDH-5 https://www.slideshare.net/slideshow/isoenzymes-59711638/59711638 https://www.biologybrain.com/isoenzymes/