Enzymes Introduction Lecture Notes PDF

27/01/2025 ENZYMES ▰ hastens chemical reactions in organic matter ▰ catalyzes single or limited num...

27/01/2025 ENZYMES ▰ hastens chemical reactions in organic matter ▰ catalyzes single or limited number of chemical reactions ENZYMOLOGY ▰ ▰ large molecules confined in cells increase membrane permeability allows them to enter the blood ▰ measured in terms of their activity and not of their absolute values ▰ appear in serum after cellular injury, degradation of cells or from storage areas ▰ used clinically as evidence of organ damage ▰ specific for substrate that it converts to a defined product 2 1 2 ENZYMES ES COMPLEX Each enzyme contains: 1. Active site ▰ water free cavity where the substrate interacts 2. Allosteric site E + S ES E+ P ▰ cavity other than the active site ▰ bind regulator molecules ▰ physical binding of a substrate to the active site of an enzyme 3 4 3 4 FACTORS 1 A. ENZYMECONCENTRATION A. ENZYME CONCENTRATION ▰ the higher enzyme concentration, the faster is the reaction FACTORS AFFECTING ENZYMATIC REACTIONS 5 enzyme concentration faster reaction 6 5 6 1  27/01/2025 FACTORS FACTORS B. B. SUBSTRATE SUBSTRATE CONCENTRATION CONCENTRATION C. COFACTORS ▰ with the amount of enzyme exceeding the amount of substrate, the 1. Coenzymes reaction rate steadily increases as more substrate is added ▰ second substrates (organic compound) ▰ saturation kinetics: when the substrate concentration reaches its maximal ▰ ↑ coenzyme = ↑ velocity of reaction value, higher concentration substrate no longer result in increase rate of ▰ essential for absolute enzymatic activity reaction ▰ ex. NAD and NADP 7 8 7 8 FACTORS FACTORS C. COFACTORS C. COFACTORS 2. Activators 3. Metalloenzymes ▰ inorganic ions ▰ inorganic ions attached to a molecule ▰ alters spatial configuration of enzyme ▰ ex. catalase and cytochrome oxidase for proper binding ▰ ex. calcium, zinc, chloride, magnesium, potassium 9 10 9 10 FACTORS ▰ the substrate concentration at which D. INHIBITORS the reaction velocity is half of the maximum level 1. Competitive Inhibitor ▰ constant for a specific enzyme and ▰ binds to active site of enzyme substrate under defined reaction ▰ substrate and inhibitor compete for condition and is an expression of the same site of enzyme relationship between the velocity of an ▰ substrate > inhibitor = reversible enzymatic reaction and substrate reaction concentration ▰ inhibitor = excess substrate ▰ has the ability to alter Km 11 Michaelis – Menten Curve / Constant 12 11 12 2  27/01/2025 FACTORS FACTORS D. INHIBITORS D. INHIBITORS 2. Non competitive Inhibitor 3. Uncompetitive Inhibitor ▰ does not compete with substrate ▰ inhibitor binds to ES complex (allosteric site) ▰ ↑ substrate = ↑ ES = ↑ inhibition ▰ irreversible inhibition ▰ enzyme + inhibitor = ↓ reaction enzyme – substrate inhibitor 13 complex 14 13 14 FACTORS FACTORS E. ISOENZYMES F. TEMPERATURE ▰ same catalytic reaction but slightly ▰ active: 25°C, 30°C, 37°C (optimum) different molecular structure ▰ denatured: 40°C to 50°C ▰ varies from amino acid sequence ▰ inactivated: 60°C to 65°C ▰ ↑ enzyme activity + isoenzyme ▰ ↑ temperature = ↑ movement of molecules = ↑ reaction ▰ temperature coefficient (Q10): ↑ 10°C = ↑ two fold in enzyme activity 15 16 15 16 FACTORS FACTORS G. HYDROGEN ION CONCENTRATION or pH H. STORAGE ▰ physiologic reactions: pH 7 to 8 ▰ low temperature (refrigeration/freezing): ▰ extreme level pH: reversibly inactive ▻ denature enzyme ▰ repeated freezing and thawing: denature ▻ influence ionic state proteins ▻ structural change ▰ –20°C: preservation for long period ▻ change in charge of amino acid ▰ 2°C to 8°C: ideal storage for substrate and residue in the active site coenzyme 17 ▰ RT: ideal storage for LD (LD4 and LD5) 18 17 18 3  27/01/2025 FACTORS 2 I. HEMOLYSIS ▰ ↑ enzyme concentration J. LACTESCENCE or MILKY SPECIMEN ▰ ↓ enzyme concentration ENZYME NOMENCLATURE 19 20 19 20 ENZYME NOMENCLATURE ENZYME NOMENCLATURE ▰ standardized by Enzyme Commission (EC) in 1961 and revised in 1972 and ENZYME ABBREVIATION IDENTIFICATION NUMBER 1978 1. Acid phosphatase ACP 3.1.3.2 ▰ classified according to: 2. Aldolase ALD 4.1.2.13 ▻ biochemical function 1st digit → classification 3. Alkaline phosphatase ALP 3.1.3.1 ▻ substrate catalyzed 2nd and 3rd digit → subclass 4. Amylase AMS 3.2.1.1 ▻ class of reaction catalyzed 4th digit → serial number 5. Alanine aminotransferase ALT 2.6.1.2 ▰ individual identification numbers (specific) 6. Aspartate aminotransferase AST 2.6.1.1 ex. AMS EC 3.2.1.1 7. Aldolase ALD 4.1.2.13 8. Angiotensin converting enzyme ACE 3.4.15.1 21 9. Creatine kinase CK 2.7.3.2 22 21 22 ENZYME NOMENCLATURE 3 ENZYME ABBREVIATION IDENTIFICATION NUMBER 10. True / Acetyl cholinesterase ACHE 3.1.1.7 11. Pseudocholinesterase PCHE 3.1.1.8 12. Gamma glutamyl transferase GGT 2.3.2.2 13. Glucose-6-phosphate G-6-PD 1.1.1.49 dehydrogenase 14. Lactic dehydrogenase LD 1.1.1.27 ENZYME 15. Leukocyte alkaline phosphatase LAP 3.4.11.1 CLASSIFICATION 16. Lipase LPS 3.1.1.3 17. 5’Nucleotidase 5’N 3.1.3.5 23 24 23 24 4  27/01/2025 ENZYME CLASSIFICATION ENZYME CLASSIFICATION CLASS FUNCTION EXAMPLE/S CLASS FUNCTION EXAMPLE/S Cytochrome oxidase (CO), Lactate catalyze hydrolysis or splitting Esterase: Acid phosphatase (ACP), dehydrogenase (LD), Malate 3. Hydrolases of a bond by the addition of Alkaline phosphatase (ALP), (CHS), catalyze the removal or 1. Oxidoreductases dehydrogenase (MDH), Isocitrate water Lipase (LPS) addition of electrons (redox) dehydrogenase (ICD), Glucose-6- catalyze removal of groups phosphate dehydrogenase (G-6-PD) Glutamate decarboxylase (), Pyruvate from substrates without 4. Lyases decarboxylase (), Tryptophan catalyze the transfer of a Creatine kinase (CK), Aspartate hydrolysis decarboxylase (), Aldolase (ALD) chemical group other than aminotransferase (AST), Alanine product contains double bond 2. Transferases hydrogen from one substrate aminotransferase (ALT), (OCT) catalyze the intramolecular to another Glucose phosphate isomerase (), 5. Isomerases arrangement of the substrate Ribose phosphate isomerase () compound 25 26 25 26 ENZYME CLASSIFICATION 4 CLASS FUNCTION EXAMPLE/S catalyze the joining of two substrate molecules coupled 6. Ligase with breaking of Synthase pyrophosphate bond in ATP or similar compound ENZYME THEORY 27 28 27 28 ENZYME THEORY 5 LOCK AND KEY THEORY ▰ Emil Fisher ▰ shape of the key (substrate) must fit into the lock (enzyme) INDUCED FIT THEORY ENZYME ▰ Kochland KINETICS ▰ substrate binding to the active site of the enzyme 29 30 29 30 5  27/01/2025 ENZYME KINETICS 6 ▰ ↑ free energy or kinetic energy on substrate + ↓ free energy or kinetic energy on product = ↑ chemical reaction ENZYME SPECIFICITY 1. Absolute specificity: combines with only one substrate and catalyzes only one reaction 2. Group specificity: combines with all the substrates in a chemical group ENZYME 3. Bond specificity: reacting with specific chemical bonds REACTIONS 31 32 31 32 ENZYME REACTION ENZYME REACTION 1. Zero order reaction: depends on enzyme concentration UNITS FOR ENZYMATIC ACTIVITY 2. First order reaction: directly proportional to substrate concentration GENERAL METHODS 1. International unit (IU or U): 1 micromole of substrate/minute 2. Katal unit (KU): 1 mole of substrate/second 1. Fixed time: reactants combined → reaction on designated time → reaction stopped → measurement 2. Continuous monitoring / Kinetic assay: multiple measurements, more preferred over fixed time 33 34 33 34 ENZYME REACTION REMEMBER! ▰ Enzymes are quantified based on their activity rather than absolute values CAUSES OF ELEVATED PLASMA ENZYME LEVELS ▻ change in substrate concentration ▻ change in product concentration 1. Impaired removal of enzyme from plasma ▻ change in coenzyme concentration 2. Increase permeability of cell membrane ▰ Units used to report enzyme levels are activity units 3. Increase in the number of cells or the production of cells ▰ Definition for activity unit must consider change in pH, temperature, 4. Increase in the normal cell turnover substrate, etc. 5. Decrease clearance of enzymes from the circulation 6. Tissue necrosis and degeneration 35 36 35 36 6  27/01/2025 THANKS! Don’t forget you’re human. It’s okay to have a meltdown. Just don’t unpack and live there. Cry it out. Then refocus on where you’re headed 37 37 7