WEEK 1.pdf

1 ENZYMES ENZYMES These are proteins produced by living cells that hastens chemical reactions in organic matter. They are measured in terms of their activity and not in terms of their absolute values. They are large molecules and they are normally confined within cells unless increased membrane perm...

1 ENZYMES ENZYMES These are proteins produced by living cells that hastens chemical reactions in organic matter. They are measured in terms of their activity and not in terms of their absolute values. They are large molecules and they are normally confined within cells unless increased membrane permeability allows them to enter the blood. They frequently appear in the serum after cellular injury, degradation of cells or from storage areas. Abnormal large amounts of enzymes in serum are used clinically as evidence of organ damage. reaction or a limited number of chemical reactions, and it iS Each enzyme catalyzes a single specific for a substrate that it converts to a defined product. Factors Affecting Enzyme Reactions: 1. Enzyme Concentration The higher the enzyme concentration, the faster is the reaction, because more enzyme is present to bind with the substrate. 2. Substrate Concentration With the amount of enzyme exceeding the amount of substrate, the reaction rate steadily increases as more substrate is added. However, when the substrate concentration reaches a maximal value, higher concentration of substrate no longer result in increased rate of reaction (saturation kinetics). 3. Cofactors Nonprotein entities that must bind to particular enzymes before a reaction occurs. A. COENZYMES organic compound (second substrates) increasing coenzyme concentration will increase the velocity of an enzymatic reaction. EX: NADP B. Activators inorganic ions (alters spatial configuration of the enzyme for proper substrate binding) EX: calcium, zinc, chloride, magnesium, K C. Metalloenzymes inorganic ion attached to a molecule (catalase). 4. Inhibitors Enzymatic reactions may not progress if an inhibitor interferes with the reaction. A. Competitive Inhibitor physically bind to the active site Of an enzyme. both the substrate and inhibitor compete for the same active site of the enzyme. with a substrate concentration significantly higher than the concentration of the inhibitor, the inhibition is reversible. the effEct of the inhibitor can be counteracted by adding excess substrate to bind the enzyme. has the ability to alter the apparent Michaelis-Menten constant (Km). dilution of serum results to reduction in the concentration of this inhibitor, thus increasing the rate of reaction. B. NONCompetitive Inhibitor binds an enzyme at a place other than the active site. do not compete with the substrate but look for areas other than the active site. because the inhibitor binds the enzyme independently from the substrate, increasing substrate concentration does not reverse the inhibition. the substrate and inhibitor (commonly metallic ion) may bind an enzyme simultaneously. the presence of the inhibitor when it is bound to the enzyme slows the rate of the reaction. C. Uncompetitive Inhibitor the inhibitor binds to the enzyme-substrate (ES) complex, so that increasing the substrate concentration results in more ES complexes to which the inhibitor binds and thereby increases the inhibition. increasing substrate concentration, increases inhibition. 5. Isoenzymes These are enzymes having the same catalytic reactions but slightly different molecular structures. The importance of the total enzyme activity is enhanced by fractionating the isoenzymes. 6. Temperature Increasing temperature usually increases the rate of a chemical reaction by increasing the movement of molecules. Enzymes are active af 25°C, 30°C, or 37°C. 37°C - optimum temperature for enzymatic activity The rate of denaturation increases as the temperature increases, and is usually significant at 40°C-50°C. 60-65°C - inactivation of enzymes refrigerated/freezing temperature- enzymes reversibly inactive Repeated freezing and thawing tends to denature proteins and should be avoided. Temperature Coefficient (Q10) there will be a two-fold increase in enzyme activity for every 10°C increase in temperature. 7. PH an enzyme or influence its ionic state resulting in Extreme pH level may denature an enzyme or structural change or change in the charge of amino acid residue in the active site. Most physiologic reactions occur in the pH range of 7-8. 8. Storage -20°C = preservation for longer period of time (enzymes) 8° C = temperature ideal for substrate and coenzymes Room temperature = ideal for storage of LDH (LD4 and LDS) 9. Hemolysis mostly increases enzyme concentration. 10. Lactescense or milky specimen decreases enzyme concentration. Enzyme Nomenclature To standardize enzyme nomenclature, the Enzyme Commission (EC) adapted a classification system in 1961 and revised the standards in 1972 and 1978. Enzymes are classified according to their biochemical functions, indicating substratE and class of reaction catalyzed, and are designated by individual identification numbers. The first diGit places the enzvme in its classifications (six classifications). The final and fourth number's, is a serial number that is specific to each enzyme in a subclass. Acid Phosphatase Alkaline Phosphatase E.C.3.1.3.2 Creatine Kinase E.C.2.7.3.2. E.C. 3.1.3.1 True/Acetyl Cholinesterase E.C.3.1.1.7 Amylase E.C.3.2.1.1 Pseudocholinesterase E.C. 3.1.1.8 Alanine Aminotransferase E.C.2.6.1.2 Gamma Glutamyl Transferase E.C.2.3.2.2 Aspartate Aminotransferase E.C.2.6.1.1 G-6-PD EC. 1.1.1.49 Aldolase E.C.4.1.2.13 Lipase E.C.3.1.1.3 Lactic Dehydrogenase E.C.1.1.1.27 5' Nucleotidase E.C.3.1.3.5 Angiotensin Converting E.C.3.4.15.1 Enzyme CLASSIFICATION OF ENZYMES 1. Oxidoreductases Catalyze the removal or addition of electrons (redox reaction). EX: CO, LDH, MDH, ICD, G-6-PD A + B: A: + B 2. Transferases Catalyze the transfer of a chemical group other than hydrogen from onE substrate to another. EX: CK, AST, ALT, OCT, GGT A + BX --> AX + B 3. Hydrolases Catalyze hydrolysis or splitting of a bond by the addition of water (hydrolytic reactions). EX: Esterases: ACP, ALP, CHS, LPS Peptidases: Trypsin, Pepsin, LAP Glycosidase: AMS, Galactosidases A + H2O --> B + C 4. Lyases Catalyze removal of groups from substrates w/o hydrolysis. The product contains double bonds. EX: Glutamate decarboxylase Pyruvate decarboxylase Aldolase Tryptophan Decarboxylase A --> B + C 5. Isomerases Catalyze the intramoleculaR arrangement of the substrate compound EX: Glucose phosphate isomerase ribose phosphate isomerase 6. Ligases Catalyze the joining of two substrate molecules, coupled with breaking of the pyrophosphate bond in ATP or similar compound. EX: Synthase A + B --> AB ENZYME STRUCTURE General Properties of Enzymes Each enzyme contains: 1. active site water-free cavity, where the substrate interacts with particular charGed amino acid residues; is a 3dimensional protein structure. 2. allosteric site cavity other than the active site; may bind regulator molecules. 1. prosthetic group coenzyme bound tightly to the enzyme 2. Apoenzyme the protein part of an enzyme The inactive form of an enzyme that requires a non-protein component to become functional 3. holoenzyme apoenzyme + prosthetic group active, catalytically functional enzyme Enzyme Theories 1. Emil Fisher's/Lock and Key Theory the shape of the key (substrate) must fit into the lock (enzyme). 2. Kochland's/Induced Fit Theory based on the substrate binding to the active site of the enzyme. Enzyme KINETICS A chemical reaction may occur spontaneously if the free energy or available kinetic energy is higher for the substrate than the product. Enzymes catalyze physiologic reactions by lowering the activation energy level that the substrate must reach for the reaction to occur. An enzyme combines with only one substrate and catalyzes only one reaction - absolute specificity. Enzymes combine with all the subsrates in a chemical group - group specificity. Enzymes reacting with specific chemical bonds bond specificity. Enzymatic Reactions 1. Zero-order reaction reaction rate depends only on enzyme concentration. 2. First-order reaction reaction rate is directly proportional to substrate concentration. METHODS OF MEASUREMENT 1. Fixed-time the reactants are combined; the reaction proceeds for a designated time. The reaction is stopped and measurement is made. 2. Continuous monitoring/kinetic assay multiple measurements of absorbance changed are made during the reaction more advantegous than fixed-time. Enzyme Activity Enzymes are measured in terms of: Change in the substrate concentration. Change in the product concentration. Change in coenzyme concentration. Units for Expressing Enzymatic Activity: International Unit (IU or U) - 1 micromole of substrate/minute Katal Unit (KU) - mole of substrate/second Enzymes are quantitated based on their activity rather than absolute values. The units used to report enzyme levels are activity units. The definition for activity unit must consider change in pH, temperature, substrate, etc. CAUSES OF INCREASED SERUM LEVELS Impaired removal of enzyme from plasma. Tissue necrosis and degeneration. Increased permeability of cell membrane. Increase in the number of cells or the production of cells.