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MLAB 2401: Clinical Chemistry Keri Brophy-Martinez

MLAB 2401: Clinical Chemistry Keri Brophy-Martinez. Enzymes: Overview. Enzymes. Functional proteins that catalyse biological reactions Involved in all essential body reactions Found in all body tissues Seen in serum following cellular injury or from degraded cells

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MLAB 2401: Clinical Chemistry Keri Brophy-Martinez

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  1. MLAB 2401: Clinical ChemistryKeri Brophy-Martinez Enzymes: Overview

  2. Enzymes • Functional proteins that catalyse biological reactions • Involved in all essential body reactions • Found in all body tissues • Seen in serum following cellular injury or from degraded cells • Decrease the amount of free energy needed to activate a specific reaction

  3. General Properties of Enzymes • Not altered or consumed during reaction • Reusable • Accelerate speed of reactions

  4. General Properties of Enzymes • Holoenzyme • Functional unit • Consists of: • Apoenzyme • Cofactor/coenzyme • Proenzyme/zymogen • Inactive enzyme Holoenzyme

  5. General Properties of Enzymes • Role • Increase reaction rates while not being consumed or altered Enzyme • Substrate Product

  6. Definitions and Related Terms • Active site • Specific area of the enzyme structure that participates in the reaction(s)/interacts with the substrate

  7. Definitions and Related Terms • Allosteric site • Non-active site • May interact with other substances resulting in overall enzyme shape change

  8. Definitions and Related Terms • Isoenzymes • Structurally different enzymes that catalyze the same reaction • Multi molecular form • Similar catalytic activity • Differing biochemical or immunological characteristics • Can detect by different electrophoresis patterns, absorption patterns, or reaction with specific antibodies

  9. Definitions and Related Terms • Cofactor • Non-protein substances required for normal enzyme activity • Types • Activator: inorganic material such as minerals • (Ca 2+, Fe2+) • Co-enzymes: organic in nature • (ATP, ADP, nicotinamide)

  10. Enzyme Kinetics • Reactions occur spontaneously if energy is available • Enzymes lower the activation energy for the chemical reactions

  11. Enzyme Kinetics • Activation energy • Excess energy that raises all molecules at a certain temperature to the activation state

  12. Enzyme Kinetics • Basic reaction • S + E ES E + P • Where • S= substrate • Substance on which the enzyme acts • E= Enzyme • ES= enzyme-substrate product • Physical binding of a substrate to the active site of enzyme • P= Product

  13. Enzyme Kinetics & Specificity • Enzymes differ in their ability to react with different substrates • Absolute specificity • Enzyme combines with only one substrate and catalyzes one reaction • Group specificity • Combine with all substrates containing a specific chemical group • Bond specificity • Enzymes specific to certain chemical bonds • Stereoisomerism • Enzymes that mainly combine with only one isomer of a particular compound

  14. Michaelis-Menten • Relationship of the reaction velocity/rate to the substrate concentration • The Michealis-Menten Constant (Km) • The substrate concentration in moles per liter when the initial velocity is ½ V max. Michaelis-Menten Curve

  15. Michaelis-Menten • First order kinetics • Rate is directly proportional to substrate concentration • Zero order kinetics • Plateau is reached • depends only on enzyme concentration

  16. Michaelis-Menten • Equation used to distinguish different kinds of inhibition • Where • V0: velocity/rate of enzymatic activity • Vmax: The maximal rate of reaction when the enzyme is saturated • Km: (constant)the substrate concentration that produces ½ of the maximal velocity • S: substrate concentration

  17. Lineweaver-Burk Plot • Adaptation of Michaelis-Menten equation • Yields a straight line

  18. Influencing Factors on Enzymatic Reactions • Substrate Concentration • Enzyme Concentration • The higher the enzyme level, the faster the reaction • pH • Most reaction occur in range of 7.0-8.0 • Changes in pH can denature an enzyme • Temperature • Most reactions performed at 37 o C • Increasing temp increases rate of reaction • Avoid high/low temps due to denaturation of enzyme • Cofactors • Influence the rate of reaction • Inhibitors • Presence can interfere with a reaction can be reversible or irreversible

  19. Types of Inhibition • Competitive • Any substance that competes with the substrate for the active binding sites on the substrate • Reversible • Non-competitive • Any substance that binds to an allosteric site • Uncompetitive • Inhibitors bind to the ES complex • No product produced

  20. Noncompetitive Inhibition Irreversible Inhibition Competitive Inhibition

  21. Types of Inhibition Uncompetitve Noncompetitive Competitive

  22. Enzyme Nomenclature • Historical • ID of individual enzymes was made using the name of the substrate that the enzyme acted upon and adding “ase” as the suffix • Modifications were often made to clarify the reaction • International Union of Biochemistry (IUB) in 1955 appointed a commission to study and make recommendations on nomenclature for standardization

  23. Enzyme Nomenclature: IUB • Components • Systematic name • Describes the nature of the reaction catalyzed • Example: alpha 1,4-glucagon-4-gluconohydrolase • Recommended name • Working or practical name • Example: amylase • Numerical code • First digit places enzyme in a class • Second and third digit represent subclass(s) of the enzyme • Fourth digit specific serial number in a subclass • Example: 3.2.1.1

  24. Enzyme Nomenclature: IUB • Standard Abbreviated name • Accompanies recommended name • Example: AMS • Common Abbreviated name • Example: AMY

  25. Enzyme Classification: General • Plasma vs. non-plasma specific enzymes • Plasma specific enzymes have a very definite/ specific function in the plasma • Plasma is the normal site of action • Concentration in plasma is greater than in most tissues • Often liver synthesized • Examples: plasmin, thrombin

  26. Enzyme Classification: General • Non-plasma specific enzymes have no known physiological function in the plasma • Some are secreted in the plasma • Increased number of this type seen with cell disruption or death

  27. Enzyme Classification • Six classes • Oxidoreductases • Involved in oxidation-reduction reactions • Examples: LDH, G6PD • Transferases • Transfer functional groups from one substrate to another • Examples: AST, ALT • Hydrolases • Catalyze the hydrolysis of various bonds • Examples: acid phophatase, lipase

  28. Enzyme Classification • Lyases • Catalyze removal of groups from substrates without hydrolysis, product has double bonds • Examples: aldolase, decarboxylase • Isomerases • Involved in molecular rearrangements • Examples: glucose phosphate isomerase • Ligases • Catabolism reactions with cleavage of ATP • Example: GSH

  29. References • Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins. • http://regentsprep.org/Regents/biology/units/homeostasis/processes.cfm • http://student.ccbcmd.edu/~gkaiser/biotutorials/proteins/fg9.html • Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson .

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