Principles of Protein Structure - H. Schultz 1982

Protein–ligand interactions
Nucleotide binding sites
Induced fit in adenylate kinase

Substrate-induced fit enhances Specificity. Many Enzymes that transfer phosphoryl groups from ATP to acceptor molecules utilize the induced-fit mechanism to increase their specificity—specifically, to prevent H₂O from acting as an alternative phosphoryl group acceptor. A classic example is adenylate kinase (Fig. 10.5), which phosphorylates H₂O at a rate 10⁵ times slower than its specific substrate, AMP.

An enzyme that conforms to the induced-fit model [686, 687] exists almost exclusively in the inactive E state, with only a tiny fraction of molecules adopting the active E' conformation. Following Jencks' hypothesis [631], the ratio [E]/[E'] can be determined directly from the phosphorylation rates of H₂O and the specific substrate. For adenylate kinase, the [E]/[E'] ratio in the absence of the substrate must be 10⁵. The binding of the specific substrate induces a conformational change in the Active Site, thereby converting the enzyme into the active E' form (Fig. 10.5).

The majority of the binding energy is expended on driving the conformational change of the enzyme. If Jencks' assumption is correct, the observed binding constant K_obs = [E' — AMP]/[E] x [AMP] = 10⁴ M^-1 is 10⁵ times smaller than the "intrinsic" binding constant K_int = [E' — AMP]/[E] ∙ [AMP]. Using the relation ΔG = —RTlnK, we find that the total Free energy of binding resulting from the interaction of AMP with the E' enzyme form (ΔG_int = —12.5 kcal/mol) is more than twice the free energy of binding derived from the observed dissociation constant (ΔG_obs = —5.3 kcal/mol). Thus, most of the binding energy is consumed in bringing about the conformational transition of the enzyme from the inactive to the active state, while the remainder manifests as the observed binding energy. The total binding energy would equal the observed binding energy if the enzyme were locked in the active E' conformation. Unfortunately, this can only be achieved in crystalline adenylate kinase under conditions where binding constants cannot be determined [665, 688].

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Fig. 10.5. Displacement of the phosphoryl-binding loop (residues 16–22) upon transition from conformation A (solid lines) to conformation B (dashed lines) in crystalline adenylate kinase [665, 688].

In conformation B, the ATP-binding site features an adenosine pocket, a wide cleft to accommodate the phosphoryl group, and an open AMP-binding site. In conformation A, the binding site is also open; however, the phosphoryl cleft is narrow, and the adenosine pocket of the AMP-binding site is closed. These data indicate that conformation B corresponds to the free enzyme prior to AMP-induced fit, whereas conformation A corresponds to the E' enzyme form following this transition.