Support Materials

•  Goal - Chapters 5 and 6:
  To know and characterize structure / function relationships of enzymes.
   
•  Objectives:
  Students will be able to...
  • identify and define nucleophiles and electrophiles.
  • recognize and write nucleophilic addition and substitution reactions.
    The reaction catalyzed by carbonic anhydrase is an example of nucleophilic addition.
    The reactions catalyzed by lipases and proteases are examples of nucleophilic substitution. How do you decide whether a nucleophilic reaction is  "addition" or "substitution"?
  • describe the role of various metal ions in enzymatic catalysis, such as Zn(II) (i.e., carbonic anhydrase, carboxypeptidase) and Ca(II) (snake venom phospholipase A₂).
  • use electron pushing arrows to show the bond breaking and bond making steps that occur during nucleophilic addition and substitution.
    Examples:
    Use arrows to show nucleophilic attack on a carbonyl substrate.
    Use arrows to show collapse of tetrahedral oxyanion intermediate into products.
  • characterize the role of H+ transfer during enzymatic catalysis.
    Enzymes make better nucleophiles: How? Give examples.
    Enzymes make better leaving groups: How? Give examples.
  • list differences between nonregulatory (Michaelis-Menten) enzymes and regulatory (allosteric) enzymes.
    Examples: regulatory enzymes show sigmoidal dependence of v_o on [S]_o, they have quaternary structure, they can be regulated by allosteric effectors (activators and inhibitors) and by covalent modification.
  • understand that the Lineweaver-Burk plot applies only to enzymes showing hyperbolic dependence of v_o on [S]_o and that it is used because V_max (and therefore K_M) is difficult to estimate from a hyperbola.
  • use a Lineweaver-Burk plot to calculate V_max from the y-intercept and K_M from the x-intercept and to express each with appropriate units.
  • understand that disease is sometimes characterized by overexpression of a specific nonregulatory enzyme and that pharmaceutical companies design drugs to selectively inhibit these enzymes.
    Example: overexpression of carbonic anhydrase in the lens of the eye can lead to glaucoma, a condition characterized by increased excess intraocular pressure.
  • understand that pharmaceutical companies often design drugs against nonregulatory enzymes that behave as a  reversible, competitive inhibitors.
  • recognize that a reversible, competitive inhibitor increases the K_M for substrate but does not affect V_max.
  • draw the effect of a reversible, competitive inhibitor on a graph of v_o vs. [S]_o or on a graph of 1/v_o vs. 1/[S]_o.
  • identify energy diagrams that correspond to tight binding of substrate (loose binding of transition state), or to tight binding of transition state (loose binding of substrate).
  • label DG° and DG°^± on an energy diagram, and know that DG° is related to the equilibrium distribution of substrates and products (thermodynamics) and that DG°^± is related to the rate of conversion of substrates to products (kinetics).
  • explain that K_M can be a measure of substrate binding affinity (how tight or loose substrate is bound) for a nonregulatory enzyme.
    Physiological concentrations of substrate for nonregulatory enzymes are often < K_M. For example, the K_M for carbonic anhydrase in the red blood cell, obtained from v_o vs. [CO₂]_o data, is about 10 mM, whereas the blood plasma concentration of CO₂ is only about 1 mM. In other words, most carbonic anhydrase molecules are not bound with CO₂ under physiological conditions. This is consistent with the idea that enzymes show weak binding of substrates and, therefore, strong binding of transition states.
  • identify for various reactions how the transition state differs from substrate in terms of shape and properties.
    For example, in the carbonic anhydrase-catalyzed reaction, the CO₂ substrate is neutral and linear, whereas the product, bicarbonate ion, is trigonal planar and has a negative charge. The transition state between substrate and product on the reaction pathway develops the shape and charge characteristics of the product, which are different than those of the substrate. The active site is designed to stabilize the properties of the transition state (tight binding of transition state) and to interact only weakly with substrate (loose binding of substrate).
    Can you explain how the properties of the transition state would differ from those of the substrate for ester or amide hydrolysis?
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