Ch.12 Enzymes

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Ch.12 Enzymes
2013-02-15 17:30:03

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  1. Enzymes
    Chemical "lubricants" that make chemical reactions go nice and easy
  2. General Properties and definitions
    • Many chemical reactions can't occur at 37 C (too cold)
    • Many chemical reactions require added heat energy
  3. Catalysts
    • Substances that lessen the amount of energy required for chemical reactions to occur.
    • Not used up in reactions.
    • Protein catalysts
    • Required for numerous metabolic processes of all cells.
    • Cells are damaged, leak out into the plasma

    Decrease the amount of energy required to activate chemical reactions.
  4. Enzyme Structure
    • 1: Amino acid sequence
    • 2: Interaction between 2 locations on the protein chain
    • 3: Folding of chains
    • 4: 2 or more separate polypeptide chains
  5. Active site
    Physical location on the enzyme molecule which interacts with the substrate molecule.
  6. Allosteric Site
    Non-active site, but which may interact with other substances to change the overall enzyme 3D shape
  7. Isoenzymes
    Structurally different enzymes (proteins) but which catalyze the same chemical reactions
  8. Cofactor
    • Non-protein substance required for normal enzyme activity.
    • Maintain enzyme 3D structure, critical for enzyme function.
  9. Two Types of cofactors:
    • Activators: Inorganic- Mg, Ca
    • Coenzyme: Organic- NADH
  10. Holoenzyme
    Enzyme + coenzyme (prosthetic group)= Active enzyme
  11. Proenzyme (Zymogen, Apoenzyme)
    Enzyme-coenzyme=Inactive enzyme
  12. Enzyme kinetics
    • Chemical reactions occur spontaneously if the energy for reactants is higher than products.
    • The amount of energy required to stimulate molecules to break their chemical bonds and form new bonds is the activation energy.
    • Increasing the temperature can generate the activation energy. Problem for living cells.
  13. Enzymes differ in their ability to react with different substrates
    • Absolute: catalyzes only 1 specific substrate
    • Group: Catalyzes reactions of a particular chemical group
    • Bond: Catalyzes reactions of particular chemical bonds
    • Steroisomerism: Catalyzes reactions of steroisomers
  14. First Order kinetics
    Enzyme activity proportional to substrate concentration.

    • As substrate concentration increases, the reaction rate increases.
    • As substrate concentration decreases, the reaction rate decreases.
    • Reaction rate is affected by substrate concentration.

    Not a good place to measure enzyme activity.
  15. Zero Order Kinetics
    • Substrate concentration is in excess.
    • Substrate "saturates" enzymes.

    • All enzyme reacts with the excess substrate.
    • Chemical reaction rate goes to max velocity.
    • Reaction rate dependent on enzyme activity.

    Enzymes are not measured in terms of concentration, but activity.

    Suited for enzyme measurement because this is where the reaction rate is dependent on the enzyme's ability to catalyze a chemical reaction.
  16. Enzyme "concentration"
    Activity is measured under Zero Order conditions because this is where the rxn rate is dependent on the work of the enzyme.
  17. Michaelis-Menten Constant
    • Numberical constant for each enzyme and substrate under defined conditions.
    • Expresses a relationship between the reaction rate and substrate concentration
  18. pH (acidity)
    • Enzymes are proteins and subject to changes in 3D structure from pH changes.
    • pH must be carefully controlled in enzymatic reactions
  19. Temperature
    • Reaction rates vary dramatically with temp changes.
    • Reactions rates may double per 10 C increase in temp.
    • Temp. must be defined and regulated for enzymatic reactions.
    • 37 C, common standardized temperatures
  20. Cofactor
    (non-protein substances needed for enzymatic activity)
    • Activators (Ca, Fe, Mg, Mn, Zn)
    • Coenzymes (Vitamins and nucleotide phosphates)
  21. Substrate Concentration
  22. Inhibitors (Substances that decrease enzyme rxn rates)
    Competitive Inhibitors
    • Substances that bind at the enzyme's active site.
    • Competes with substrate for the active site.
    • Addition of additional substrate increases the reaction rate.
  23. Non-Competitive Inhibitors
    • Substances that bind at an enzyme's non-active site
    • Enzymes 3D shape is altered, decreasing enzyme activity
    • May bind substrate, but additional substrate has no effect on rxn rate
  24. Uncompetitive Inhibitors
    Substances that bind with enzyme-substrate complex (rare)
  25. Measuring enzyme activity
    • Enzymes not directly measured, measured in terms of catalytic activity.
    • Activity is proportional to concentration.
  26. Enzyme activity can be tested by measuring:
    • Increase of product
    • Decrease of substrate
    • Decrease of co-enzyme
    • Increase of altered co-enzyme
  27. Coupled enzyme reactions
    • Some enzyme testing utlizes other enzymatic reactions in a sequence of reactions.
    • These other accesory enzymes are not being measured.
    • Auxillary enzymes are used as reagents and added in excess so they do not become rate limiting factors in the overall process.

    All enzymatic reactions in a coupled assay must be performed at zero order kinetic.
  28. Fixed Time Assay (2 point)
    • Zero Order Kinetics
    • Substrate concentration is measured at set timed intervals to determine enzyme activity.
    • A slight delay from the beginning of the reaction to max velocity is the lag phase.
  29. Substrate depletion
    Extreme elevations in enzyme activity may deplete the substrate between measured intervals

    Cause falsely decreased activity results
  30. Multipoint Continuous Monitoring
    • Continuous measurements of substrate-product concentration are recorded by the spectrophotometer of an automated analyzer.
    • Substrate depletion is detected because the analyzer is always "looking" and will see any sudden dramatic changes