Common Features of Allosteric Proteins

Allosteric proteins are composed of multiple subunits and are regulated by both homo-tropic (substrate)and heterotropic ligands. In the case of heterotropic effects, this is usually a binding site quite different from that of the substrates or functional ligands of the protein, and may involve a separate subunit or domain of the overall protein. With homotropic effects, the spatially separate site is another chemically identical active site in the oligomer. The substrate affinity of allosteric proteins is cooperative in nature—the binding of substrate to one subunit affects the substrate binding affinity of other subunits. Cooperativity may be either positive or negative and is indicated experimentally by a curved ligand binding plot of protein activity versus substrate concentration (for comparison, a straight line is observed for non-allosteric proteins) (Fig. 9.6). A concave downwards curve (plot) indicates positive interactions/coopera-tivity, while a concave upwards plot indicates negative interactions/cooperativity. In an alternative analysis of the data, where the enzyme velocity is measured (Lineweaver-Burk plot) or the saturation with ligand is deter mined (Klotz Plot), a double-reciprocal representation (Fig. 9.7 of 1/Rate (or l/[Bound Ligand] vs. l/[Substrate] or l/[Free Ligand]) which is linear for an enzyme showing no alio steric effects, is concave downward for nega tive cooperativity or concave upward for posi tive cooperativity. A coefficient of 1 indicates no cooperativity, whereas n > 1 equals positive cooperativity and n < 1 equals negative cooperativity.

While either ligand binding or kinetic ob servations are often used to invoke allosteri< interactions, it is critically important torecog-nize that such manifestations of cooperativt interactions are not unique. Kinetic plotr showing either "positive" or "negative" coop. erativity can simply be a manifestation of £ complex kinetic mechanism, whereas binding studies indicative of "negative cooperativity' can result from multiple independent species capable of binding the same ligand with differ. ent affinities. Sigmoidal saturation curves o: binding data, however, can be attributed onlj to "positive" cooperative effects. As has beer extensively discussed (12-14), a clear demon.

of an allosteric effect requires demon. stration of an induced conformational change

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1/[Free ligand] or 1/[Substrate]

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1/[Free ligand] or 1/[Substrate]

Figure 9.7. Lineweaver-Burk or Klotz plots of data indicative of (a) negative cooperativity, where at least two distinct linear regions are seen (asshown here), or (b) positive cooperativity.

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1/[Free ligand] or 1/[Substrate]

Figure 9.7. Lineweaver-Burk or Klotz plots of data indicative of (a) negative cooperativity, where at least two distinct linear regions are seen (asshown here), or (b) positive cooperativity.

affecting a distant site (for the Koshland/ Dalziel-type models) or the demonstration of a préexistent equilibrium between R and T states of the protein for Monod-type models.

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