Anion binding coupling

Bivalent inhibitors of thrombin have been synthesized to bind the anion-binding exosite and active (catalytic) site of thrombin simultaneously. By coupling the carboxy terminal fragment of hirudin to a tripeptide (D-Phe-Pro-Arg) by including a spacer molecule, both the anion exosite and the catalytic site are blocked. An example of such a molecule is Hirulog, which has 20 amino acids and has a Kj of 2 nM (61). Its ability to block the active site has been questioned, since thrombin has been shown to cleave the Arg-Pro bond of Hirulog slowly in vivo (58). In addition to hirudin and hirudin-like compounds, three other classes of site-directed thrombin inhibitors deserve mention. 

The coupling between iron and anion binding is a critical component of transferrin function. In the absence of anion, transferrins bind Fe " weakly and nonspecifically. The putative anion site identified crystal-lographically is in an electrostatically positive region, adjacent to the side chain of an arginine and the amino terminus of an a helix. The anion site does not bridge the two domains, and so does not function directly as a latch that closes the cleft around the iron. It is possible that the anion may partially compensate for the presence of several basic residues in the cleft that enhance iron binding. 

Both the coupling of proton uptake to anion binding and the surprising low-temperature structural instability of the active site complex may mimic abstraction of the hydroxylic proton from a coordinated substrate molecule during turnover. Ionization of a coordinated alcohol by a base in the active site would be an important step for substrate activation in the catalytic mechanism. The nonturnover reactions serve as models for this process, giving us clearer insight into the catalytic reaction by mapping out an intrinsic proton transfer coordinate in the active site. 

Spin echo measurements on metal complexes have been reviewed extensively by Mims and Peisach [267]. One use of this technique has been to study anion binding at paramagnetic metal centers. When cupric ion is substituted for ferric ion in transferrin, a pattern corresponding to the weak coupling for in C-doped bicarbonate indicates that bicarbonate is bound directly to the metal ion [269]. Thus, a modulation pattern detected by spin echo can confirm that adducts with a weakly coupled nuclear spin are bound directly to the metal site. 

Density functional theory is a successful approach for the description of ground-state properties of metals, semiconductors, and insulators. It also has become an attractive method to calculate complex materials, such as proteins and carbon nanotubes. For example, DFT calculation has been used to compute anion-binding properties of 2,6-diamidopyridine dipyrromethane hybrid macrocycles (18), to predict drug resistance of HIV-1 reverse transcriptase to nevirapine through point mutations (19), and to analyze the 32-adrenergic G protein-coupled receptor (20). A free DFT software program also is available... 

When a redox-active transition metal is used as the signalling imit of a receptor, anion binding is coupled to electron transfer, i.e. anion binding changes the redox potential (couple) of the transition metal. This electrochemical shift can be represented as A, the difference in redox potentials between the receptor anion complex and the receptor alone. Concomitantly, electron transfer at the redox centre also changes the affinity of the receptor for the guest species. These coupled processes are linked thermodynamically by Eq. 1, where Kred and Kox are the stability constants of the reduced and oxidised forms of the receptor anion complex respectively [7]. 

Anion binding stabilises the oxidised form of the receptor, hence Kqx/ JCred > 1 and the redox potential of the reporter group is shifted to a more negative value (cathodic shift). The Kox/K ed ratio is also a measure of how efficient the coupling is between the metal-based reporter group and the anion-binding site. 

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