Enzymes PDF

ENZYMES Asia Taha, PhD. Ahmed Ragaa, BPharm, PhD. Michael Atef Fawzy, PhD.  INTRODUCTION: All reactions in the body are mediated by enzymes- protein catalysts that increase the rate of reactions without themselves being changed in the overall process Catalyst: It i...

ENZYMES Asia Taha, PhD. Ahmed Ragaa, BPharm, PhD. Michael Atef Fawzy, PhD.  INTRODUCTION: All reactions in the body are mediated by enzymes- protein catalysts that increase the rate of reactions without themselves being changed in the overall process Catalyst: It is a substance which accelerates the chemical reaction without consumed in the reaction Enzymes: They are protein catalysts that increase the velocity of a chemical reaction and are not consumed during the reaction They posses the following properties. -Thermolabile -Organic colloidal catalysts -Protein in nature -Produced by the living cells -Specific in their actions Item Nature comparison Enzyme Proteins Non-proteins Catalyst Chemical structure Organic Inorganic Production By living cells By chemical reactions (biological substances) (non-biological substances) Specificity Highly specific in their Non specific actions Effect of temp. Thermolabile (denaturable) Thermostable (not denaturable) Activity High catalytic efficiency Low catalytic efficiency Examples Pepsin, lipse, …….ect Copper, zinc,…….ect  Structure of enzymes: Enzymes are protein Core have the following sites: 1. Active sites: a) Catalytic site: -It is the region on the enzyme surface that catalyzes the chemical reaction, It may be separated from the substrate-binding site by a large or a small distance or they may be combined into one site. b) Substrate- binding site: -It is that at which substrate specifically binds. The catalytic site and/or substrate binding-site may be rigid or flexible. -In the rigid model, they have rigid tertiary structure. So the substrate must have a complementary shape and size in order to fit in the catalytic site. This is described as the lock and key model. -In the flexible model, the substrate induces a conformational change in the enzyme tertiary structure to fit the substrate. This is described as the induced fitting model.  c) Allosteric site: -It is the site to which an allosteric effectors is non- covalently bind -Allosteric effectors: are substances of low molecular weight having little or no structural similarity to substrate. -It causes a conformational change in the enzyme particularly at the active site(s) that decreases or increases the enzyme activity. --Thus, allosteric effectors are called negative effectors (or feed back inhibitor) if it decreases the enzyme activity. -The allosteric effectors are called positive effectors (or feed back activator) if it increases the enzyme activity.  2. Structural components of the enzyme system (holoenzyme) Some enzymes do not need any additional components to show full activity. But certain enzymes require helpers called coenzymes, cofactors or prosthetic groups to help them in achieving their action. COFACTORS AND COENZYMES  MECHANISM OF ACTION: Enzyme (substrate binding site) + Substrate (AB) → Enzyme-substrate Complex → Products (A + B) + free Enzyme (substrate binding site) again → and so on  HOW DO ENZYMES WORK? Substrates need a large amount of energy to reach a transition state, which then decays into products. The enzyme stabilizes the transition state, reducing the energy needed to form products.  Energy decreases (under catalysis) P Reaction direction EP ENERGY CHANGE T = Transition state EST ST ES S Energy required (no catalysis)  Features of Active Site…… Why energy required to reach transition state is lower in the active site? It is a magic pocket + (1) Stabilizes transition (2) Expels water CoE (1) (2) (3) Reactive groups (4) - (4) Coenzyme helps (3)  Substrate Transition state Product X If enzyme just binds substrate then there will be no further reaction Enzyme not only recognizes substrate, but also induces the formation of transition state Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252  NAMING OF ENZYMES Nomenclature of Enzymes: Each enzyme is assigned two names. A-Recommended name: It is the short and most commonly used enzyme names) a) Some have the suffix"-ase“ attached to the substrate of the reaction, Examples: glucosidase, urease, sucrase, b) Some used to describe the action performed, Examples: lactate dehydrogenase and adenylate cyclase. c)Some enzymes retain their original trivial names, which give no hint of the associated enzymatic reaction, Examples: trypsin and pepsin.  Naming of Enzymes contd B. The International Union of Biochemistry and Molecular Biology have developed a nomenclature for enzymes, the EC numbers; each enzyme is described by a sequence of four numbers preceded by "EC". The first number classifies the enzyme based on its mechanism. The top-level classification is: EC 1 Oxidoreductases: catalyze oxidation/ reduction reactions. EC 2 Transferases: transfer a functional group (e.g. a methyl or phosphate group). EC 3 Hydrolases: catalyze the hydrolysis of various bonds. EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation. EC 5 Isomerases: catalyze isomerization changes within a single molecule. EC 6 Ligases: join two molecules with covalent bonds e.g ATP:glucose phosphotransferase EC 2.7.1.1.  Classification of enzymes: A) according to their action: 1.Lyases 2.hydrolase 3.Ligase B) According to their substrate 1.Uricase 2.Arginase 3.histadase C) According to the chemical reaction 1.Reductase 2.Oxidase 3.Transaminase D) According to classes on which they act 1.Proteases 2.Lipases 3.Glycosilase E) According to induction and repression: 1.Inducible: rate of synthesis and/or activity of the enzyme is increased by inducer 2. Constitutive: Enzyme whose rate of synthesis in a cell is constant and does not depend on an inducer  MECHANISM OF ENZYME ACTION Enzymes are usually very specific as to which reactions they catalyze and the substrates that are involved in these reactions. Complementary shape, charge and hydrophilic/hydrophobic characteristics of enzymes and substrates are responsible for this specificity. Two models were proposed for enzyme action: 1. Lock and Key Model 2. Induced Fit Model  1. Lock and key model ▪ Enzymes are very specific, because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as "the lock and key" model. ▪ However, while this model explains enzyme specificity, it fails to explain the stabilization of the transition state that enzymes achieve.  2. Induced fit model Since enzymes are rather flexible structures, the active site is continually reshaped by interactions with the substrate as the substrate interacts with the enzyme. As a result, the substrate does not simply bind to a rigid active site. In some cases, such as glycosidases, the substrate molecule also changes shape slightly as it enters the active site.  2. Induced fit model Induced to transition state Lock and Key model X Not possible  ENZYME KINETICS Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products. The enzyme (E) binds a substrate (S) and produces a product (P). In 1902 Victor Henri proposed the idea that enzyme reactions occur in two stages. In the first, the substrate binds reversibly to the enzyme, forming the enzyme-substrate complex. This is sometimes called the Michaelis complex. The enzyme then catalyzes the chemical step in the reaction and releases the product.  Vmax, Km and Kcat Vmax: It is the maximum velocity (Vmax) of the enzyme. Vmax is extrapolated from the plot, At this stage all the enzyme active sites are bound to substrate. Km, : is the substrate concentration required for an enzyme to reach one-half its maximum velocity. Each enzyme has a characteristic Km for a given substrate. kcat : is the number of substrate molecules handled by one active site per second. So the efficiency of an enzyme = kcat/Km  Importance of Km: 1.Substrates are usually present in physiological fluids at a concentration around the Km values. 2.It also reflects the presence or absence of an enzyme inhibitor. 3.Km is a characteristic constant for every enzyme. 4.Km reflects the affinity of the enzyme for the substrate. The lower the Km value, the higher the enzyme affinity to substrate and the more activity of the enzyme. The higher Km reflects a low affinity of the enzyme for substrate. 5.It also reflects the effect of allostric regulators on the rate of the reaction.  Example: Hexokinase is more active than glucokinase because the amount of glucose (substrate) needed to reach 1/2Vmax in case of hexokinase is less than in case of glucokinase, i.e. Km of hexokinase is less than glucokinase. This difference is of almost physiological importance. Hexokinase or Glucokinase Glucose + ATP Glucose-6-phosphate + ADP Glucose concentration  Enzymes Specificity: Definition: Enzymes are highly specific, interacting with one or a few specific substrates and catalyzing only one type of chemical reaction. Types: There are 5 types of substrate specificity: i) Stereo specificity ii)Absolute specificity iii)Dual specificity iv)Relative specificity v) Structural specificity. i. Absolute Specificity: The enzyme acts on only one substrate, e.g., uricase enzyme acts on uric acid, arginase enzyme acts on arginine, urease enzyme acts on urea, carbonic anhydrase enzyme acts on carbonic acid ii. Stereo-specificity: the enzyme is specific to a specific isomer of a substrate and does not act on other isomers, e.g. L-amino acid oxidase acting on L-aminoacids only and D- aminoacid oxidase acting on D-aminoacids only. iii. Dual specificity: There are 2 types of dual specificity: An enzyme acting on 2 different substrates but catalyzes one type of reaction, e.g., xanthine oxidase acting on hypoxanthine and xanthine causes oxidation of both substrates into uric acid. An enzyme acting on one substrate but catalyzes 2 different reactions, e.g., isocitrate dehydrogenase acts on isocitrate causing dehydrogenation and decarboxylation iv. Relative specificity: The enzyme acts on a group of compounds related to each other in having the same type of bond and also this enzyme catalyzes the same type of reaction, e.g., Lipase catalyzes the process of hydrolysis of ester linkage present in triglycerides containing different types of fatty acids. v. Structural specificity: The enzyme is specific to the bond like the relative specificity but it requires chemical groups or atoms around this bond. Pepsin hydrolyzes the middle or terminal peptide linkages formed by the amino groups of phenylalanine or tyrosine. Trypsin attacks the peptide linkage containing the carboxyl group of arginine or lysine