Inhibitor binding binary complexes

The catalytic subunit of cAPK contains two domains connected by a peptide linker. ATP binds in a deep cleft between the two domains. Presently, crystal structures showed cAPK in three different conformations, (1) in a closed conformation in the ternary complex with ATP or other tight-binding ligands and a peptide inhibitor PKI(5-24), (2) in an intermediate conformation in the binary complex with adenosine, and (3) in an open conformation in the binary complex of mammalian cAPK with PKI(5-24). Fig.l shows a superposition of the three protein kinase configurations to visualize the type of conformational movement. 

As we have just seen, the initial encounter complex between an enzyme and its substrate is characterized by a reversible equilibrium between the binary complex and the free forms of enzyme and substrate. Hence the binary complex is stabilized through a variety of noncovalent interactions between the substrate and enzyme molecules. Likewise the majority of pharmacologically relevant enzyme inhibitors, which we will encounter in subsequent chapters, bind to their enzyme targets through a combination of noncovalent interactions. Some of the more important of these noncovalent forces for interactions between proteins (e.g., enzymes) and ligands (e.g., substrates, cofactors, and reversible inhibitors) include electrostatic interactions, hydrogen bonds, hydrophobic forces, and van der Waals forces (Copeland, 2000). 

Figure 6.17 Cartoon depicting the interactions of FKBP with inhibitor and the subsequent binding of the FKBP Inhibitor binary complex to the enzyme calcineurin (E).

Often high-affinity, or tight binding, interactions with enzymes is the result of a very slow dissociation rate of the enzyme-inhibitor binary complex. 

Imidazole also acts as a substrate-competitive inhibitor, forming both binary complexes with LADH, and ternary complexes in the presence of coenzyme. X-Ray studies show that imidazole also binds to the. catalytic zinc by displacing the water molecule.1361 The presence of imidazole at the active site also enhances the rate of carboxymethylation14658 of Cys-46 with both iodoacetate and iodoacetamide.1420 This enhancement of alkylation has become known as the promotion effect .1421 Imidazole promotion also improves the specificity of the alkylation.1422 Since Cys-46 is thought to be alkylated as a metal-thiol complex, imidazole, on binding the active site metal, could enhance the reactivity by donating a electrons to the metal atom, which distributes the increased electron density further to the other ligands in the coordination sphere. The increased nucleophilicity of the sulfur results in promoted alkylation.1409... 

The crystal structure of the E. colt DHFR/MTX binary complex (9) reveals that the inhibitor binds in the active site in a kinked fashion (Figure 8). Asp-27 forms a salt-bridge to the bound pteridine ring while Phe-31 allows for the bend in the bound inhibitor. The remainder of the active site cavity surrounding the pterin ring is composed of amino acid residues that create a very hydrophobic environment. 

Kinetic studies of reversible inhibition by substrate analogs give evidence of the mode of action of the inhibitor and the types of enzyme-inhibitor complex formed, and estimates of their dissociation constants. The complexes may be isolated and sometimes crystallized. Studies of the stabilities, optical properties, and structures of ternary complexes of enzymes, coenzymes, and substrate analog in particular, as stable models of the catalytically active ternary complexes or of the transition state for hydride transfer (61,79,109,115-117), can only be touched upon here there is direct evidence with several enzymes that the binding of coenzymes is firmer in such complexes than in their binary complexes (85,93,118), which supports the indirect, kinetic evidence already mentioned for a similar stabilization in active ternary complexes. 

However, the findings (171) that binary complexes of zinc-free enzyme and coenzyme bind substrates and substrate competitive inhibitors such as isobutyramide cannot be taken as evidence that zinc does not participate in the catalytic action. Several SH groups are probably oxidized in the zinc-free enzyme (170). Evidence has also been presented (172) of other structural differences compared to the catalytically active enzyme. Thus, artificial binding to this enzyme with no relevance to the catalytic action is not unlikely. 

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