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Are the Common Allosteric Models Correct?

Are the Common Allosteric Models Correct?

A review of a lecture given by Dr. Gregory Reinhart, Texas A&M University
Webpage designed by Biochemistry 107 Group 2:
  • Kevin Attenhofer
  • Stephanie Michinock
  • Samantha Pohl
  • James Grieme
  • Charles Delashmit

    A Review of Enzyme Reactions:

  • most enzymes are proteins although some nucleic acids (ribozymes) have catalytic properties
  • enzymes accelerate rates of reaction without being changed by the reaction
  • enzymes accelerate rates of reaction without changing the equilibrium position of the reaction
  • typically enzymes carry out a chemical reaction, such as the transformation of substrates (reactants) to products, A-->B, through an enzyme-substrate complex
  • rates are accelerated 10^3 to 10^17-times compared to the uncatalyzed rate
  • enzymes are highly specific for their substrates: they are stereospecific for binding of reactant and stereoselective for the formation of the correct stereoisomer of the product

  • the substrate is the reactant in an enzyme catalyzed reaction. A ligand is a general term for any substance that binds to a protein. Substrates are ligands that bind to proteins and undergo a specific chemical reaction
  • the substrate binds to the enzyme in a very specific manner at the active site
  • the active site is a cleft or pocket in the 3-D structure of a protein, often between subunits or domains
  • there is a complementary fit between the shape of the active site and the shape of substrate - stereospecificity between the shape of the active site and the shape of substrate
  • this allows formation of specific non-covalent bonds between active site and substrate
  • a good analogy is the specificity between a lock and key, although because the 3-D structure of proteins and enzymes is flexible, the binding of substrate to enzyme can and does induce a conformational change in the protein and the transition state in the substrate

    Allosteric: "other site"

  • allosteric enzymes are enzymes that have at least 2 binding sites:
    • active site for reactants
    • regulatory site
  • allosteric inhibitor: small molecule which attaches to an allosteric enzyme at its regulatory site and inactivates it
  • allosteric activator: small molecules which attaches to an allosteric enzyme and activates it, thus increasing rates of enzymatic reaction

    For our purposes, allosteric refers to the regulation (either inhibition or activation) of enzyme activity due to the binding of a metabolite to a site on the enzyme that is different from the active site. An allosteric inhibitor binds to an allosteric site and shifts the conformational equilibrium to one that is relatively inactive. We will be refering to inhibitors because the contrast in their perception is much more drastic than that of activators.

    Common Model:

    It is commonly thought that most allosteric effects can be explained by either the concerted (MWC) model put forth by Monod, Wyman, and Changeux, or by the sequential model described by Koshland, Nemethy, and Filmer. Both postulate that enzyme subunits exist in one of two conformations, tensed (T) or relaxed (R), and that relaxed subunits bind substrate more readily than those in the tense state. The two models differ most in their assumptions about subunit interaction.

    Concerted Model: The concerted model of allostery postulates that enzyme subunits are connected in such a way that a conformational change in one subunit is necessarily conferred to all other subunits. Thus all subunits must exist in the same conformation. To summarize:

  • all subunits must exist in the same conformation
  • when one sub-unit bonds, the entire enzyme becomes inhibited

    Sequential Model: The sequential model of allostery holds that subunits are not connected in such a way that a conformational change in one induces a similar change in the others. Thus all enzyme subunits need not exist in the same conformation. To summarize:

  • subunits need not exist in the same conformation
  • molecules of substrate bind via induced fit protocol
  • the inhibition only occurs on the sub-unit that is bonded

    Goal

    Disprove the accuracy of the commonly accepted allosteric models.

    Predictions

    Concerted - inhibition is the same regardlesss of the position of the bonded sub-unit.
    Sequential - inhibition is almost maximal in one position; minimal in the others.

    Experiment

    Phosphofructokinase
    Fru-6-P + MgATP --> MgADP + Fru-1,6-BP
    From: E. Coli
    Composition: homotetramer

    • 1 active site and 1 allosteric site per sub-unit
    • 4 sub-units
    Regulation: PEP inhibits / MgADP activates

    Adding PEP acts like an inhibitor in that Fru-6-P isn't allowed to bond as tightly.

    A computer-generated image of phosphofructokinase.

    This is the same picture as the previous image, but only the parts of the enzyme relative to Dr. Reinhart's experiment are shown. Yellow structures represent active sites on the enzyme. Red structures represent allosteric sites.

    The purpose of the experiment is to determine if the individual allosteric sites (x-axis) have an effect on the bonding of a specific active site.

    Through manipulation, all but one active site and one allosteric site were completely inhibited. The pattern of inhibition was rotated four times so that each allosteric site had a chance to opperate along with the chosen active site. At the same time, negative markers were added to the molecule so it could later be determined from other hybrids.

    The negative markers were used to determine which of the samples were the 1:3 hybrid.

    In the following four images, arrows are used to indicate (via a numerical representation as well as relative thickness of the arrow) the impact the bonding of each allosteric site has on the active site. First is the Common Model approach. Following that are Dr. Reinhart's results in varying pH solutions (indicated at the upper left corner of the image).

    Conclusion

    Multiple allosteric ligands contribute a corresponding number of couplings that are:

  • unique in magnitude (not concerted)
  • additive in their contribution (not sequential)