Enzyme Chemistry Lecture Notes PDF

Lippincott’s illustrated reviews Chapter 5, Page 53 Lectures 7 - 8 Enzyme Chemistry 1  Specific Objectives By the end of this lecture students can be able to: Know the function of coenzyme, apoenzyme and holoenzyme Differentiate between competi...

Lippincott’s illustrated reviews Chapter 5, Page 53 Lectures 7 - 8 Enzyme Chemistry 1  Specific Objectives By the end of this lecture students can be able to: Know the function of coenzyme, apoenzyme and holoenzyme Differentiate between competitive and noncompetitive inhibition Recognize the role of competitive inhibition in the action of statin drugs. Explain the role of enzyme inhibitors in the synthesis of antibiotics. Know how enzyme activity can be regulated. 2  enzymeProtine Enzymes Are protein catalyst that increase the rate of reactions and decrease the energy of the reaction without being change in the overall process. Enzyme-catalyzed reactions are highly efficient, proceeding from 103-10 e 8 times faster than uncatalyzed reactions. i 3 All reactions in the body are mediated by enzymes. Lack of enzymes will lead to block in metabolic pathways causing inborn errors of metabolism. The substance upon which an enzyme acts, is called the substrate. The enzyme will convert the substrate into the product or products. E Turnover number is the number of molecules of substrate converted to product per enzyme molecule per 4 second.  Nomenclature of enzymes Each enzyme is assigned two names. The first is short, recommended name, convenient for every day use. The second is the more complete, systematic name, which is used when an enzyme must be identified without ambiguity. 5  Classification of enzymes According to its function Class 1. Oxidoreductase: Transfer of hydrogen, catalyze oxidation-reduction reaction as conversion of lactate to pyruvate by lactate dehydrogenase. Class 2. Transferase: Catalyze transfer of C-, N-, or P- containing groups. Such as conversion of serine to glycine by serine hydroxy methyl transferase 6 Class 3. Hydrolases: Catalyze cleavage of bonds by addition of water, e.g. Urease convert urea to CO2 and NH3 Class 4. Lyases: Cleave without adding water, catalyze cleavage of C-C, C-S and certain C-N bonds, such as cleavage of pyruvate by pyruvate decarboxylase into acetaldehyde. 7 Class 5. Isomerases: Catalyze racemization of optical or geometric isomers, such as conversion of methylmalonyl CoA to Succinyl CoA by methylmalonyl CoA mutase. Class 6. Ligases: ATP dependent condensation of two molecules, e.g. acetyl CoA carboxylase. It catalyze formation of bonds between carbon and O, S, N 8 Note: Some RNAs can act like enzymes, usually catalyzing the cleavage and synthesis of phosphodiester bonds. RNAs with catalytic activity are called ribozymes, and are much less commonly encountered than protein catalysts. 9 Holoenzymes: The term holoenzyme refers to the active enzyme with its nonprotein component. The term apoenzyme is inactive enzyme without its nonprotein part. If the nonprotein part is a metal ion such as Zn 2+ or Fe2+, it is called a cofactor. If it is a small organic molecule, it is termed a coenzyme. 10 Coenzymes that only transiently associate with the enzyme are called co-substrates. If the coenzyme is permanently associated with the enzyme and returned to its original form, it is called a prosthetic group as FAD. Coenzymes frequently are derived from vitamins. Example, NAD+ contains niacin and FAD contains riboflavin. 11  Coenzyme is a low molecular weight organic substance, without which the enzyme cannot exhibit any reaction. Function of coenzyme and cofactor The coenzyme and cofactor are essential for the 12 biological activity of the enzyme.  Cofactor Is a metallic ions when present in some enzymes show higher activity Example, calcium will activate lipase. 13 Active sites: Enzyme molecules contain a special pocket or cleft called the active site. The active site contains amino acid chains that participate in substrate binding and catalysis. It is called catalytic residues or catalytic groups. As example Proteolytic enzymes having a serine residue at the active center called serine proteases. The specific substrate bound to the active site. 14 The substrate binds the enzyme, forming an enzyme-substrate (ES) complex. Binding is thought to cause a conformational change in the enzyme (induced fit) that allows catalysis. ES is converted to an enzyme-product (EP) complex that subsequently dissociates to enzyme and product. 15 16  Michaelis-Menten Equation A. Reaction model: This also called enzyme-substrate complex theory. The enzyme (E) combines with the substrate (S), to form an enzyme- substrate (ES) complex, which immediately breaks down to the enzyme and the product (P). k1 k2 E + S ↔ E-S complex → E+P k-1 Where, K1, k-1 and k2 are rate constants. 17 B. Michaelis-Menten Equation: Vmax [S] V0 = -------------------- Km + [S] Where, V0 = initial reaction velocity Vmax = maximal velocity Km = Michaelis constant = (k-1 + k2)/k1 [S] = substrate concentration Vmax It is the maximum rate of reaction, it is the rate of reaction when the enzyme is saturated with substrate. The relationship between rate of reaction and concentration of substrate depends on the affinity of the enzyme for its substrate. Km It is the concentration of substrate which permits the enzyme to achieve 18 half Vmax. 19  Factors influencing enzyme activity 1- Enzyme concentration: Velocity of reaction is increased proportionately with the concentration of enzyme, when substrate concentration is unlimited. 20 2- Substrate concentration: As substrate concentration is increased, the velocity is also correspondingly increased in the initial phases; but the curve flattens afterwards. The maximum velocity thus obtained is called Vmax or point of saturation. 21 3- Concentration of products: When product concentration is increased, the reaction is slowed, stopped or even reversed. 22 4. Temperature: 1. Increase of velocity with temperature 2. Decrease of velocity with higher temperature 23 5. Effect of pH: Each enzyme has an optimum pH, on both sides of which the velocity will be drastically reduced. The pH decides the charge on the amino acid residues at the active site. Usually enzymes have the optimum pH between 6 and 8. Some important exceptions are Pepsin (with optimum pH 1- 2), alkaline phosphatase (optimum pH 9-10) and acid phosphatase (4-5). 24 25  Inhibition of enzyme activity Inhibitor is any substance that can diminish the velocity of an enzyme-catalyzed reaction. In general, irriversible inhibitors bind to enzyme through covalent bonds. Reversible inhibitors typically bind to enzyme through noncovalent bonds. The most common inhibitions are either competitive or noncompetitive. 26 A. Competitive inhibition: In this type, the inhibitor binds to the same active site of the enzyme because inhibitor is similar in its structure to substrate. 27 The inhibitor molecules are competing with the normal substrate molecules for attaching with the active site of the enzyme. E + S → E-S → E + P E + I → E-I If substrate concentration is enormously high when compared to inhibitor, then the inhibition is reversed. 28 For examples, Succinate dehydrogenase reaction is inhibited by malonate, which are structural analogs of succinate. Dihydropteroate synthase (enzyme in prokaryotes involved in the synthesis of folic acid needed for DNA synthesis) is inhibited by sulphonamide (antibacterial agent) which is structural similar to PABA. Dihydrofolate reductase is inhbited by methotrexate (anticancer agent) which is structural similar to folic acid. 29  Statin drugs as examples of competitive inhibitors This group of antihyperlipidemic agents inhibits the first committed step in cholesterol synthesis. This reaction is catalyzed by hydroxymethylglutaryl -CoA reductase (HMG-CoA reductase). 30 Statin drug such as atorvastatin (Lipitor) and pravastrate (Pravachol) are structural analogue of the natural substrate for this enzyme, and compete effectively to inhibit HMG-CoA reductase. By doing so, they inhibit de novo cholesterol synthesis, thereby lowering plasma cholesterol levels. 31 B- Noncompetitive inhibition This occur when the inhibitor and substrate bind to different site on the enzyme. 32 An increase in the substrate concentration generally does not relieve this inhibition. A variety of poisons, such as iodoacetate, heavy metal ions (silver, mercury) and oxidizing agents act as irreversible noncompetitive inhibitors. 33 Examples for noncompetitive inhibition: 1. Lead forms covalent bond with sulfhydryl side chains of cysteine in protein. Ferrochelatase, an enzyme that catalysis insertion of Fe2+ into protoporphyrin (a precurser of heme) is an example of enzyme sensitive to inhibition by lead. 34 35 2. Insecticides, whose neurotoxic effects are a result of their covalent binding to acetylcholine esterase (enzyme that catalyze cleaves of neurotransmitter, acetylcholine. 36  Enzyme Inhibitors as Drugs At least half of ten most commonly dispensed drugs in United States act as enzyme inhibitors. 1. The widely prescribed β-lactan antibiotics, such as penicillin and amoxicillin, act by inhibiting enzymes involved in bacterial cell wall synthesis. 37 2. Drugs may also act by inhibiting extracellular reactions. This is illustrated by angiotensin-converting enzyme (ACE) inhibitors. 38 They lower blood pressure by blocking the enzyme that cleaves angiotensin I to form the potent vasoconstrictor, angiotensin II. These drugs, which include captopril, enalapril, and lisinopril, cause vasodilation and a resultant reduction in blood pressure. 39  Regulation of Enzyme Activity A. Regulation of allosteric enzymes Allosteric enzymes are regulated by molecules called effectors (also called modifiers) that bind noncovalently at a site other than the active site. Function of allosteric effector: 1. Alter the affinity of the enzyme for its substrate. 2. Modify the maximal catalytic activity of the enzyme. Negative effectors : effectors that inhibit enzyme activity. Positive 40 effectors: effector that increase enzyme activity. 41 1. Homotropic effectors: When the substrate itself serves as an effector, the effect is said to be homotropic. Most often, an allosteric substrate functions as a positive effector. 2. Heterotropic effectors: The effector may be different from the substrate, in which case the effect is said to be heterotropic. Heterotropic effectors are commonly encountered, for example, the glycolytic enzyme phosphofructokinase-1 is allosterically inhibited by citrate, which is not a substrate for the enzyme 42 B. Regulation of enzymes by covalent modification Many enzymes may be regulated by covalent modification, most frequently by the addition or removal of groups such as peptide chain or phosphate groups 43 1. Phosphorylation and dephosphorylation: It is addition or removal of phosphate groups from specific serine, threonine, or tyrosine residues of the enzyme. Protein phosphorylation is recognized as one of the primary ways in which cellular processes are regulated. 44 Phosphorylation reactions are catalyzed by a family of enzymes called protein kinases that use adenosine triphosphate (ATP) as a phosphate donor. Dephosphorylation reaction: Phosphate groups are cleaved from phosphorylated enzymes by the action of phosphoprotein phosphatases. 45 2. Partial proteolysis: Another type of activation by covalent modification is the conversion of an inactive proenzyme or zymogen to the active enzyme. Example, splitting a single peptide bond, and removal of a small polypeptide from trypsinogen, the active trypsin is formed. This results in unmasking of the active center. Note: All the gastrointestinal enzymes are synthesized in the form of proenzymes, and only after secretion into the alimentary canal, they are activated. 46 This prevents autolysis of cellular structural proteins. C. Induction and repression of enzyme synthesis Cells can also regulate the amount of enzyme present by altering the rate of enzyme degradation or the rate of enzyme synthesis. The increase (induction) or decrease (repression) of enzyme synthesis leads to an alteration in the total population of active sites. For example, elevated levels of insulin as a result of high blood glucose levels cause an increase in the synthesis of key enzymes involved in glucose metabolism 47 48 Reference Book: Champe, P. C., Harvey, R. A. and Ferrier, D. R., 2005. Biochemistry “Lippincott’s Illustrated Reviews”, 5th or 6th Edition 49 Reference Book: Vasudevan, D. M., Sreekumari, S., and Kannan, V.., 2011. Textbook of biochemistry for medical students, 6th Edition. 50