Enzymes PDF

ENZYMES DR. AHMED ABDULGHANY Ass. Professor. of Medical Biochemistry Head of Biochemistry Department  Objectives ▪ Definition, Properties, active site, and structure of enzymes ▪ Regulation of enzyme activity ▪ Plasma enzymes and isoenzymes ▪ Factors affecting enzyme activities ▪ Enzyme i...

ENZYMES DR. AHMED ABDULGHANY Ass. Professor. of Medical Biochemistry Head of Biochemistry Department  Objectives ▪ Definition, Properties, active site, and structure of enzymes ▪ Regulation of enzyme activity ▪ Plasma enzymes and isoenzymes ▪ Factors affecting enzyme activities ▪ Enzyme inhibition ▪ Regulation of enzymes  Definition of enzyme Enzymes are biological catalysts that increases the rate of a chemical reaction without being itself changed. They are found in all tissues and fluids of the body. GENERAL PROPERTIES OF ENZYMES 1. They are protein in nature 2. They are needed in small amounts. 3. They are highly specific; i.e. each enzyme acts on a specific substrate 4. They increase reaction rates without affecting its equilibrium  Cofactors Enzymes are classified, according to their chemical composition into simple and conjugated (complex) enzymes I. Simple enzymes: They are composed of amino acids only. II. Complex (conjugated) enzymes: formed of protein part and II. Conjugated Enzymes (Holoenzymes) non protein part (cofactor) ▪ Coenzyme binds loosely Protein part Non-protein part ▪ Prosthetic group binds firmly to the enzyme. (apoenzyme) Prosthetic group Coenzyme ENZYME NOMENCLATURE ▪ First part is the name of the substrates for the enzyme. ▪ Second part is the type of reaction catalyzed by the enzyme. ▪ This part ends with the suffix “ase”. ▪ Example: Lactate dehydrogenase ▪ Sometimes the name gives no idea of source, function or reaction catalyzed by the enzyme. Example: trypsin, thrombin, pepsin  Zymogens ▪They are inactive enzymes because their catalytic sites are masked by a polypeptide chain. ▪They are activated by cleavage of this polypeptide chain; e.g. pepsinogen is inactive, whereas Pepsin is active. MECHANISM OF ENZYME ACTION Two models have been proposed to explain the enzyme action 1)The Lock and Key Theory Substrat fits its binding site Like a key fits into a lock ▪ This explains enzyme specificity ▪ Also explains the loss of activity when enzymes denature  2) Induced Fit Model The substrate and active site change shapes to accommodate each other Interaction of the substrate with the enzyme induces a conformational changes of the enzyme in the formation of complementary binding site This explains the enzymes that can react with a range of substrates of similar types Active Sites of Enzymes 1. Catalytic site: ▪ It is the main site on the enzyme at which the substrate binds 2. Allosteric site: ▪ It is another specific site on the enzyme away from the catalytic site that binds the effector. ▪ This effector may stimulate (positive) or inhibit (negative) the reaction. Classification of Enzymatic Reactions 1. Oxidoreductases: They catalyze an oxidation – reduction reaction between two substances, also called oxidases,oxygenases, hydroxylases, dehydrogenases, or reductases. 2. Transferases: They catalyze the transfer of a group other than hydrogen from one substance to another. Example: phosphotransferases (Kinases) 3. Hydrolases: They catalyze hydrolysis i.e. breakdown of a chemical bond by adding H2O. example, lipases Classification of Enzymatic Reactions 4-Lyases: They catalyze simple splitting of the substrate without adding H2O. examples, (Decarboxylases) they remove CO2. 5-Ligases: join molecules together. Use ATP as an energy source e.g. Synthetases 6-Isomerses: They catalyze the interconvertion of one isomer into the other e.g. phosphohexose isomerase. They also include the following types: Epimerases and Mutases Isoenzymes or Isozymes ▪ They are different forms of the same enzyme that catalyze the same reaction, use the same coenzyme and the same substrate. They have ▪ Different Km & Vmax ▪ Different physical properties ▪ Different immunological reactions  ▪ Example -lactate dehydrogenase (LDH) It is a tetrameric protein made of two types of subunits namely H and M It exists as 5 different isoenzymes with various combinations of H and M subunits LDH-I is elevated in myocardial infarction. Creatine Kinase (has 3 isoenzymes) ▪ It is a dimmer, i.e. contains 2 subunits; B & M ▪ CK BB increases in brain infarction. ▪ CK MB increases in myocardial infarction. ▪ CK MM increases in muscle diseases. PLASMA ENZYMES I.Functional plasma enzymes These are enzymes normally present in blood to perform certain physiological functions. Examples: ▪ Proenzymes of blood clotting, prothrombin, …. ▪ Lipoproteinlipase. ▪ Pseudocholine esterase. October 14, 2024 II. Non functional enzymes ▪ These enzymes are produced and act inside the cells ▪ They are present in a very low concentration in blood ▪ They increase in blood in cases of tissue damage. ▪ Examples: 1. Liver Transaminases: ALT & AST 2. Pancreatic amylase and Lipase, 3. Alkaline phosphatase & Acid phosphatase 4. Lactate dehydrogenase [LDH] 5. Creatine phosphokinase [CPK] Enzyme kinetics ▪ The rate (velocity) of a reaction: is the change of concentration of the reactants or products per unit time. ▪ The initial velocity(vi ): is the velocity measured when very little substrate has reacted. It is dependent on both substrate and enzyme concentration. ▪ The maximum velocity (vmax) : is the velocity obtained when all enzymes molecules are saturated with substrate. Michaelis-Menten kinetics When substrate concentration is blotted against reaction velocity the curve would be hyperbola This equation describes how reaction velocity varies with substrate concentration. Leonor Michaelis 1875-1949  Km ▪ Km (Michaelis constant)= The substrate concentration that produces half maximum velocity (½ Vmax) ▪ When exactly half the enzyme molecules are saturated with (S) the velocity is half maximal velocity (½ Vmax) ▪ Km is a characteristic of an enzyme and reflects the affinity of the enzyme for that substrate. ▪ Small Km: reflects a high affinity of the enzyme for substrate ▪ Large Km: reflects a low affinity of the enzyme for substrate Lineweaver – Burk plot Michaelis-Menten equation has been rearranged by Lineweaver – Burk Vmax x [S] ▪ By invert this equation to be: Vi Km + [S] 1 Km 1 [S] Vi x + Vmax [S] Vmax [S] 1 Km 1 1 Vi x + Vmax [S] Vmax ▪ This equation represents a straight line ▪ Equation is particularly important to determine the effect of drugs on enzymes  Enzyme Inhibition A- Competitive inhibition ▪ There is structural similarity between inhibitor and substrate ▪ The inhibitor binds at the same active site as the substrate ▪ Increase substrate concentration will release the inhibitor ▪ May be reversible or irreversible Effect of competitive inhibtion on V max and Km This competitive inhibitor does not affect V max but increase Km of substrate.  B- Non – competitive inhibition ▪ There is no structural similarity between Inhibitor and substrate ▪ Inhibitor and substrate bind to different sites on enzyme ▪ Inhibitor can bind either free Enz or Enz-S-complex Effect of Non- competitive inhibition on V max and Km ▪ It decreases V max, but It does not affect Km. r 1 i to i b V h i In ve it i p et 1 m co V2max Non + hi b itor No in 1 - Km 1 V1max 0 1 [S] C- Uncompetitive inhibitors ▪ Uncompetitive inhibitors also do not resemble the substrate and bind to a different site on the enzyme. However, in contrast to noncompetitive inhibitors, they can only bind to the enzyme-substrate complex, not the enzyme alone. ▪ It decreases both the V max and Km October 14, 2024 October 14, 2024 October 14, 2024  Enzyme Regulation Long term Short term Regulation of enzyme Regulation of enzyme synthesis degradation Allosteric Induction Covalent modification Repression  Regulation of enzyme Activity A. Long Term Regulation: by controlling the enzyme quantity (it may take hours or days) ▪ Quantity of an enzyme is determined by: 1. Controlling rate of enzyme synthesis: ▪ The synthesis of an enzyme from DNA is controlled by: ▪ Induction or ▪ Repression 2. Controlling rate of enzyme degradation: Enzyme degradation involves hydrolysis of the enzyme Induction and Repression a. Induction: ▪ Increase the rate of enzyme synthesis at the gene level ▪ Inducers: they are molecules that increases the rate of enzyme synthesis at the gene level, they may be the substrate of the enzyme or it may be a hormone b. Repression: ▪ Inhibition of the enzyme synthesis at the gene level ▪ Repressors: They may be a product of metabolic pathways which inhibit enzyme synthesis. Repressor also may be a hormone B- Short term regulation Short term regulation: These types of regulation can rapidly change the already exciting enzyme from an inactive form to a fully active form and vice versa. 1. Allosteric activation and inhibition 2. Reversible covalent modifications  1-Allosteric Regulation ▪ Some molecules called modifiers, modulators bind to regulatory enzymes at allosteric site (site other than the active site). ▪ Binding induces a conformational change that affects the affinity of the enzyme to the substrate ▪ Binding is non-covalent (reversible) ▪ They may increase the enzyme activity : (+ve) modulators (allosteric activator) ▪ Or they may decrease enzyme activity: (-ve) modulators (allosteric inhibitor)  Feed back inhibition ▪ It is the inhibition of an enzyme in a metabolic pathway by the end product of that pathway 2- Covalent modification (hormonal regulation) ▪ Phosphorylation/dephosphorylation ▪ Reversible Phosphorylation of the enzyme occurs by kinase which transfers a phosphate group from ATP to a serine/threonine amino acids in the enzyme ▪ Dephosphorylation of the enzyme occurs by phosphatase ▪ The activity of kinases and phsophatases is under hormonal and neural control ▪ Glugagon hormone causes phosphorylation ▪ Insulin hormone causes dephosphorylation ▪ Phosphorylation activates some enzymes and inactivates others FACTORS AFFECTING ENZYME ACTIVITY Will discussed in the lab  Potentially confusing enzyme nomenclature ▪ Synthetase (requires ATP) ▪ Synthase (no ATP required) ▪ kinase is an enzyme that catalyzes the transfer of phosphate groups from high-energy phosphate-donating molecules to specific substrates. ▪ Phosphatase (uses water to remove phosphate group) (dephosphorylation) ▪ Phosphorylase (which catalyze the addition of inorganic phosphate groups to an acceptor) ▪ Dehydrogenase (e.g. NAD+/FAD is an electron acceptor in a redox reaction) ▪ Oxidase (O2 is the acceptor, and oxygen atoms are not incorporated into substrate) ▪ Oxygenase (one or both oxygen atoms are incorporated).  REFERENCES ▪Lippincott’s Illustrated Reviews: ▪Biochemistry. ▪Eighth Edition ▪Chapter:5 October 14, 2024 Questions and answers October 14, 2024 Thank You