Enzymes PDF

CHAPTER 4 | ENZYMES BIOCHEMISTRY | LECTURE Three Important Characteristics of Enzymes Lipases can act on lipids 1. They have Enormous Catalytic Power (hydrolyzation) 2. They are highly specific in the reactions they...

CHAPTER 4 | ENZYMES BIOCHEMISTRY | LECTURE Three Important Characteristics of Enzymes Lipases can act on lipids 1. They have Enormous Catalytic Power (hydrolyzation) 2. They are highly specific in the reactions they Phosphatases act on any molecule catalyze with phosphate esters 3. their activity as catalysts Can be Regulated (Phosphodiester bond) 3. Stereochemical specific Catalytic Efficiency super absolute specific Catalytic Power it will act only on one specific refers to the acceleration of the reaction rate Isomer/enantiomer (L-form over that for the noncatalyzed reaction only/D-form only) most specific of all Catalysts increase the rate of chemical reaction without Regulation being used up in the process 1. Compartmentalization it is not permanently changed, and may be are not present everywhere in the cell used over and over some enzyme are only present on the Enzymes lower the activation energy of a following: reaction, allowing it to achieve equilibrium ○ Cytoplasm more rapidly ○ Mitochondria ○ Endoplasmic Reticulum Carbonic anhydrase 2. Gene Control enzyme that speed up the removal of CO2 there are genetic in-born regulator out of the body inside the body cell it combines CO2 with H2O to form carbonic they regulate certain Acid (H2CO3) enzyme—gene-coded enzymes 1 molecule of Carbonic anhydrase can act on 36 Million molecules of CO2 per minute note: there should be something that would stop enzyme catalysts functioning if there's enough Specificity substrate to avoid excess product. enzymes act on specific substrate or reactant Urease Nomenclature and Classification act only on urea Enzyme Commission (EC) 1. substrate Subtype of Specificity 2. functional group 1. Absolute specificity 3. type of reaction catalyzes the reaction of only one 4. + ase substance/substrate 2nd most specific EC Group Name Type of Reaction Example: Code Catalyzed Urease that act only on urea EC 1 Oxidoreductases Oxidation-reduction rxns 2. Relative Specificity catalyzes the reaction of specific EC 2 Transferases Transfer of functional family of the Substrate group Examples: EC 3 Hydrolases Hydrolysis rxns Proteases (collective term) that can act on Peptides EC 4 Lyases addition to =bonds/ v.v. / cleavage of =bonds 1  Covalent Bond EC 5 Isomerases Isomerization rxns EC 6 Ligases Formation of bonds W/ Cofactor ATP cleavage if the non-protein component is weakly Example: bound, and easily separated from the rest of the protein Non-covalent Bond It can be an organic substance (Coenzyme) or may also be an inorganic ion (usually a metal cation: Mg2+, Zn2+, or Fe2+. Substrates for the following enzymes Coenzymes cofactor that has an organic substance Enzymes Substrate derived from the different vitamin Maltase Maltose ○ Fat soluble: Vit ADEK ○ Water soluble: Ascorbic Acid & Vit B Peptidase Peptide NAD+ Glucose-6-phosphate Glucose-6-phosphate nicotinamide adenine dinucleotide isomerase nicotinamide form a Nicotinic Acid/Niacin (Vitamin B3) from vitamin Nicotinamide General Enzyme Names and the Reaction that It is a dinucleotide they Catalyze Three Parts of Dinucleotide: Enzyme Reaction Catalyzed ○ Sugar Component Decarboxylase Removal of carboxyl group ○ purine or pyrimidine-nitrogenous from compounds base ○ phosphate group Phosphatase Hydrolysis of phosphate ester linkages The Mechanism of Enzyme Action Peptidase Hydrolysis of peptide How do enzymes work? linkages by interacting with a substrate, and a Esterase Formation of ester substrate binding to a particular site linkages Active site is where the substrate binds to the enzyme Enzyme Cofactors Allosteric site where noncompetitive inhibitor binds reversibly to the enzyme at a a conjugated proteins function only in the site other than active site presence of specific non protein molecules or When the substrate binds to the active site metal ions called prosthetic group via some combination of intermolecular Coenzymes forces, it will form a product played vital role in metabolic reactions Enzyme-substrate (ES) Complex. The final organic molecules that accompanied main product will be the regeneration of original enzymes enzymes and formation of the product They can add or remove protons or electrons True Prosthetic Groups if the non-protein component is tightly bond, and forms an integral part of enzyme Sucrase structure present in the microvilli of small intestine 2  responsible for splitting sucrose into 1 glucose and 1 fructose Examples of Enzyme Turnover Number Carbonic anhydrase 36,000,000 Lock-and-Key Theory CO2 + H20 -> H2CO3 explain the high specificity of enzyme activity Catalase Enzyme surfaces accommodate substrates 5,600,000 having specific shapes and sizes so only 2H202-2H20 + O2 specific substances “fit” in an active site to Cholinesterase form an ES complex. 1,500,000 Limitation of this Theory is that it requires hydrolysis of acetylcholine enzyme conformations to be rigid. Penicillinase by Emil Fischner enzymes that can destroy penicillin For specific enzyme Beta-lactamase Induced-Fit Theory 1 molecule can destroy 120,000 molecules of modification of the lock-and-key theory penicillin proposes that enzymes have flexible conformations that may adapt to incoming Factors that Affect Enzyme Activity substrates. 1. Enzyme Concentration Active site adopts a shape that is The concentration of an enzyme, [E], complementary to the substrate only after is typically low compared to that of the substrate is bound. the substrate. Increasing [E] also for relative-specific enzymes increases the rate of the reaction: more [E] gives more [ES]; Enzyme Activity reversible reaction refers to the catalytic ability of an enzyme to The rate of the reaction is directly increase the rate of reaction proportional to the concentration of Two ways to Measure the enzyme (doubling [E] doubles the International Unit rate of the reaction), thus, a graph of defines enzyme activity as the amount of reaction rate vs. enzyme enzyme that will convert a specified amount concentration is a straight line of substrate to a product within a certain time 2. Substrate Concentration used by doctors, physicians, etc. on Increasing [S] increases the rate of laboratory result the reaction, but eventually, the rate Example: Amylase - enzyme for acute reaches a maximum (vmax), and pancreatitis remains constant after that. Example: SGPT - enzyme from the liver The maximum rate (vmax) is reached (drug-induced hepatitis—0-40 units) when the enzyme is saturated with substrate, and cannot react any faster under those conditions. Turnover Number number of substrate acted on by one 3. Temperature molecule of the enzyme per minute most enzyme will work between Example: 1 carbonic anhydrase can act on 36 0-40°C million molecules of CO2 most optimum range is 25-40°C more common number are closer to 1000 molecules per minute 3  Like all reactions, the rate of rapidly-acting, highly toxic inhibitor, enzyme-catalyzed reactions increases which interferes with the with temperature. iron-containing enzyme cytochrome Because enzymes are proteins, oxidase beyond a certain temperature, the ○ Cell respiration stops, and death enzyme denatures. occurs within minutes. Every enzyme-catalyzed reaction has ○ An antidote for cyanide poisoning an optimum temperature at which the (cyanide kit) has three components. enzyme activity is highest, usually One of it is sodium thiosulfate, which from 25º-40ºC; above or below that converts cyanide into thiocyanate value, the rate is lower. (less poisonous), which does not bind to cytochrome 4. Effect of pH ○ Hydroxocobalamin - acts faster Raising or lowering the pH influences than cyanide kit the acidic and basic side chains in ○ Cyanocobalamin (Vit B12) - enzymes. Many enzymes are also resulting product, which is not denatured by pH extremes. (E.g., poisoning pickling in acetic acid [vinegar] preserves food by deactivating Heavy Metal bacterial enzymes.) ○ Heavy metal poisoning results when optimum pH of most enzymes at pH 7 mercury or lead ions bind to —SH or near pH 7 will increase rate of (thiols/sulfhydryl) groups on enzymes. reaction upto optimum pH after that Heavy metals can also cause protein it will result to a decline denaturation. Pb and Hg can cause (denaturation) (enzyme are inactive) permanent neurological damage. pH of Stomach: 1.5-3.5 ○ Heavy-metal poisoning is treated by administering chelating agents Enzyme-Inhibition (chelators: EDTA in lead poisoning, An enzyme inhibitor is a substance that DMSA, penicillamine, etc.) which bind decreases the rate of an enzyme-catalyzed tightly to metal ions, allowing them to reaction. be excreted in the urine. Many poisons and medicines inhibit one or ○ Edta will attract the Lead (Pb) and it more enzymes and thereby decrease the rate will release the calcium and Sodium: of the reactions they carry out. NaCaEDTA 2- + Pb2 -> PbEDTA 2- + Some substances normally found in cells Ca + Na inhibit specific enzymes, providing a means for internal regulation of cell metabolism. Antibiotics are enzyme inhibitors that act on life 1. Irreversible Inhibition processes that are essential to certain occurs when an inhibitor forms a strains of bacteria. covalent (permanent) bond with a Sulfa Drugs specific functional group of an Gerhard Domagk, 1935; Nobel enzyme and will lead to inactivation Prize 1939 of enzyme 1st Antibiotics that discovers Examples: Penicillins Cyanide ion, (CN-) Alexander Fleming, 1928; ○ irreversible inhibitor of an enzyme Nobel Prize, 1945 Cytochrome Oxidase C. It is a 4  Interfere with transpeptidase, which bacteria use in the construction of cell walls. 2. Reversible Inhibitor binds reversibly to an enzyme, Competitive inhibition can be establishing an equilibrium between reversed by either increasing the the bound and unbound inhibitor concentration of the substrate, or non-covalent bonding decreasing the concentration of the Once the inhibitor combines with the enzyme, in accordance with Le enzyme, the active site is blocked, Châtelier’s principle. and no further catalysis takes place. The inhibitor can be removed from B. Noncompetitive Inhibitor the enzyme by shifting the bind reversibly to the enzyme at the equilibrium. “allosteric site”, a site other than the There are two types of reversible active site, changing the 3D shape of inhibitors: competitive and the enzyme and the active site, so noncompetitive. that the normal substrate no longer A. Competitive Inhibitor fits correctly. bind to the enzyme’s active site and Noncompetitive inhibitors do not look compete with the normal substrate like the enzyme substrates. molecules. They often have structures Increasing the substrate that are similar to those of the normal concentration does not affect substrate. noncompetitive inhibition because it can’t bind to the site occupied by the inhibitor. Example: Sulfa drugs - inhibit the formation of folic acid - similar structure with PABA which The Regulation of Enzyme Activity bacteria need to build folic acid in Enzyme Regulation order to grow. Enzymes work together to facilitate all the - Sulfanilamide blocks PABA from biochemical reactions needed for a living fitting into the active site of the organism. To respond to changing conditions enzyme which builds folic acid, and cellular needs, enzyme activity requires causing the bacteria to eventually die. very sensitive controls: Since humans obtain folic acid from their diet rather than by a. activation of zymogens manufacturing it, it is not harmful to b. allosteric regulation the patient. c. genetic control In competitive inhibition, there are Activation of Zymogens two equilibria taking place: 5  Zymogens or proenzymes are inactive When isoleucine builds up, it binds to the precursors of an enzyme. allosteric site on threonine deaminase, Some enzymes in their active form would changing its conformation so that threonine degrade the internal structures of the cell. binds poorly. This slows the reaction down so These enzymes are synthesized and stored as that the isoleucine concentration starts to inactive precursors, and when the enzyme is fall. needed, the zymogen is released and When the isoleucine concentration gets too activated where it is needed. low, the enzyme becomes more active again, Activation usually requires the cleavage of and more isoleucine is synthesized one or more peptide bonds. The digestive enzymes pepsin, trypsin, and chymotrypsin, as well as enzymes involved in blood clotting, are activated this way. Genetic Control The synthesis of all proteins and enzymes is under genetic control by nucleic acids. Increasing the number of enzymes molecules present through genetic mechanisms is one way to increase production of needed products. Enzyme induction occurs when enzymes are Allosteric Regulation synthesized in response to cell need. Compounds that alter enzymes by changing This kind of genetic control allows an the 3D conformation of the enzyme are called organism to adapt to environmental changes. modulators. The coupling of genetic control and allosteric They may increase the activity (activators) or regulation allows for very tight control of decrease the activity (inhibitors). cellular processes. (Non-competitive inhibitors are examples of β-galactosidase is an enzyme in the this activity.) bacterium Escherichia coli that catalyzes the Enzymes with quaternary structures with hydrolysis of lactose to D-galactose and binding sites for modulators are called D-glucose. allosteric enzymes. These variable-rate enzymes are often located at key control points in cell processes. In the absence of lactose in the growth Feedback inhibition occurs when the end medium, there are very few -galactosidase product of a sequence of enzyme-catalyzed molecules. reactions inhibits an earlier step in the ○ In the presence of a process. This allows the concentration of the lactose-containing medium, product to be maintained within very narrow thousands of molecules of enzyme limits.The synthesis of isoleucine from are produced. threonine is an example of allosteric ○ If lactose is removed, the production regulation. of th-e enzyme once again decreases. Threonine deaminase, which acts in the first step of the conversion pathway, is inhibited by the isoleucine product. Medical Application of Enzymes Enzymes in Clinical Diagnosis 6  ○ Some enzymes are found exclusively in tissue cells. If they are found in the bloodstream, it indicates damage to that tissue; the extent of cell damage can sometimes be estimated from the magnitude of serum concentration increase above normal levels. ○ The measurement of enzyme concentrations in blood serum is a major diagnostic tool, especially in Each type of tissue has a distinct diseases of the heart, liver, pancreas, pattern of isoenzyme percentages. prostate, and bones. Serum levels of LDH can be used in the diagnosis of a wide range of diseases, such as anemias involving the rupture of red blood cells, acute liver diseases, congestive heart failure, and muscular diseases such as muscular dystrophy. Elevated levels of LDH₁ and LDH₂ indicate myocardial infarction Isoenzymes Elevated levels of LDH₅ indicate ○ Isoenzymes are slightly different possible liver damage. forms of the same enzyme produced by different tissues. Although all note: 2 main control: Allosteric l Regulation & Gene forms of a particular isoenzyme Control catalyze the same reaction, their structures are slightly different and their location within body tissues may vary. ○ The enzyme lactate dehydrogenase (LDH) has a quaternary structure that consists of four subunits of two different types: H — main subunit found in heart muscle cells. M — main form in other muscle cells. ○ There are five possible ways to combine these subunits to form the enzyme (see next slide); each of these forms has slightly different properties, which allow them to be separated and identified. 7