Biochemistry of Enzymes and Clinical Enzymology Lecture Notes 2021-2022 PDF

CBFblock 2021-2022 Lecture 9,10 Biochemistry and clinical enzymology Prof Naglaa Kamal Idriss Professor of Medical Biochemistry Objectives Define enzymes & describe structural components of the enzyme system. Discuss mechanism of enzyme action Explain types of enzyme sp...

CBFblock 2021-2022 Lecture 9,10 Biochemistry and clinical enzymology Prof Naglaa Kamal Idriss Professor of Medical Biochemistry Objectives Define enzymes & describe structural components of the enzyme system. Discuss mechanism of enzyme action Explain types of enzyme specificity Enumerate factors affecting enzyme activity Describe kinetics of enzyme reaction  What is an enzyme? Enzymes are biological catalysts Functional (or active) sites in the enzymes system: in gaps Enzymes are composed of long chains of amino acids that have folded into a very specific three-dimensional shape which contains an active sites. a) Substrate binding-site. b) Catalytic site. c) Allosteric site. a) Substrate-binding site: In the rigid model, they do not change their shape after combination with substrate. the lock and key model. In the flexible model, the substrate induces a conformational change in the enzyme to fit the substrate. This is described as the induced fitting model. b) Allosteric site: 1. The term allosteric means change in shape”. 2. The allosteric site is usually far from the catalytic site(s)  Substrate specificity of enzymes 1. Absolute Specificity: The enzyme acts on only one substrate Uricase enzyme acts on uric acid 2. Dual specificity: 2 types 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 into -ketoglutarate. 3-Stereo-specificity: the enzyme is specific to a specific isomer only of a substrate. L-amino acid oxidase acting on L-AAs only and D- amino acid oxidase acting on D-AAs only.. 4- Relative specificity: enzyme acts on a group of compounds having the same type of bond e.g Lipase catalyzes the hydrolysis of ester linkage present in triglycerides & Amylase catalyzes the hydrolysis of glycosidic linkages present in starch. 5. Structural specificity: group specificity The enzyme is specific to the bond but it requires chemical groups around this bond. e.g. Trypsin attacks the peptide linkage containing the carboxyl group of arginine or lysine.  Enzyme kinetics The presence of enzymes lowers the energy required for the reaction to occur ( activation energy).  Enzyme kinetics terminology [S] – substrate concentration Vo – initial velocity of a reaction. Vmax – maximal velocity of a reaction. Km – Michaelis-Menten equation The concentration of substrate at which the rate of the reaction is half-maximal Significance of Km value: Km reflects the affinity of the enzyme for the substrate.  Michaelis-Menten equation By increasing the substrate concentration [S], the value of the reaction(Km) velocity increases until reaching a plateau, where the velocity is maximum (Vmax) due to the occupational of all the enzyme molecules.  Factors affecting the rate of enzyme catalyzed reaction( Regulation of enzyme activity 1- Temperature: increase temperature, increase the enzyme activity. (37C-40C). 2- PH: There is optimal PH at which the enzyme acts maximally act at (7.4) 3-Concentration of the enzyme: Increase the enzyme concentration leads to increase the rate of enzyme reaction 4- Concentration of the substrate Leads to increase the rate of enzyme catalyzed reaction. 5-Protecting the enzymes from light and radiation which lead to their denaturation. 6- Protecting the enzymes from oxidation or inhibitors. 7-Concentration of cofactors or coenzymes: Causes an increase in the rate of the enzyme action. 8- Effect of end products or feedback regulation  Enzyme inhibitors Enzyme inhibitors are substances that inhibit the enzyme activity and divided into. 1. Competitive enzyme inhibitors:- The inhibitor is structurally similar to the substrate and binds to substrate binding site on the enzyme surface in competition with the substrate. Examples: Allopurinol is a competitive inhibitor for xanthine oxidase that synthesizes uric acid from purines. Therefore, allopurinol is used for treating Gout. Methotrexate is competitive inhibitor of dihydrofolate reductase that convert dihydrofolate to tetrahydrofolate which is essential for cell division, used as anticancer agent. 2- Non-competitive enzyme inhibitors:- The inhibitor has no structural similarity to the substrate and doesn't bind to substrate binding site on enzyme surface but binds to different site on it.  Zymogen Cleavage(or proenzymes): Definition: Most enzymes are synthesized in an inactive form called zymogens or proenzymes) Its significance is: To protect the secretory cells and transporting duct system. To keep the enzyme in a storable. To provide mechanisms for regulating the enzymes activity.  Zymogens are activated by: Release of inhibitory polypeptide by cleavage or conformational change: Association with a cofactor e.g Mg2+. Association with another protein  Isozyme (isoenzyme)  Isoenzymes (isozymes) are multiple forms of the enzyme that have the same catalytic activity.  They are physically distinct and differ in electrophoretic mobility and liability to inhibitors.  Iso means the same and isoenzyme means the same enzyme  Example of isoenzymes Many enzymes are present in isoenzyme form: 1. Lactate dehydrogenase 2. Creatine kinase 3. Acid phosphatase 4. Alkaline phosphatase  Lactate dehydrogenase (LDH) It is an enzyme that catalyzes the removal of 2 hydrogen atoms from lactic acid forming pyruvic acid. Lactate dehydrogenase Lactic acid Pyruvic acid NAD NADH+H  LACTATE DEHYDROGENASE (LDH)  It is a tetrameric protein and made of two types of subunits namely H = Heart, M = skeletal muscle  there are 5 physically distinct isoenzymes of this enzyme with various combinations of H and M subunits, known as LDH-1, LDH-2, LDH-3, LDH-4 and LDH-5.  Its level in plasma increases in: . Myocardial infarction (heart diseases). . Viral hepatitis (liver disease). . Leukaemia (blood disease) Isoenzy Composi Present Elevated me tion in in name LDH1 ( H4) Myocardiu myocardial m, RBC infarction LDH2 (H3M1) Myocardiu m, RBC LDH3 (H2M2) Kidney, brain LDH4 (H1M3) Liver, Skeletal muscle LDH5 (M4) Liver, Skeletal Skeletal muscle and muscle liver diseases Clinical significance: -Damage to either of myocardium or liver causes increase in total serum LDH and the type of specific isozyme is easily identifiable. -In normal serum, LDH2 (H3M) is the most prominent isozyme, and LDH-5 is rarely seen. - After myocardial infarction, LDH-1 and LDH-2 predominate - In acute viral hepatitis, LDH-5 and LDH-4 predominate. - In malignancy:- Total serum LDH is frequently elevated in neoplastic diseases. there is increase in LDH-3, LDH-4 and LDH-5. An increase in LDH-5 is seen in breast carcinoma  CREATINE KINASE (CK) Creatine kinase is a dimer made of 2 monomers“M” (for muscle) and “B” (for brain). Three different isoenzymes are formed:CPK- 1, CPK-2, CPK-3.  ALKALINE PHOSPHATASE (ALP)  High levels of ALP is indicative of extrahepatic obstruction rather than intrahepatic obstruction  In bones, the enzyme is derived from osteoblasts. Hence increased in bone diseases like rickets, osteomalacia, neoplastic diseases with bone metastates. References  Sanchez, Henry et al. USMLE Step 1 Pathology Lecture Notes. [New York, N.Y.]: Kaplan Medical,2021. Print. Le, Tao et al. First Aid For The® USMLE.  Thieme Test Prep for the USMLE®: Medical Biochemistry Q&A 1st Edition by Joseph D. Fontes (Author), Darla McCarthy (Author)  Enzymes: Biochemistry, Biotechnology, Clinical Chemistry 2nd Edition by T Palmer (Author), P L Bonner (Author)