SECONDARY INTERACTIONS SIGNIFICANTLY REMOVED FROM THE SULFONAMIDE BINDING POCKET OF CARBONIC-ANHYDRASE-II INFLUENCE INHIBITOR BINDING CONSTANTS

A series of competitive inhibitors of carbonic anhydrase II (CAII; EC 4.2.1.1) that consists of oligo(ethylene glycol) units attached to p-benzenesulfonamides with pendant amino acids, H(2)NSO(2)C(6)H(4)CONHCH(2)CH(2)OCH(2)CH(2)OCH(2)CH(2)NHCOCHRNH(3)(+), have been synthesized and examined using competitive fluorescence assays. Three of the strongest inhibitors, designated EG(3)NH(3)(+), EG(3)GlyNH(3)(+), and EG(3)PheNH(3)(+), have been studied by X-ray crystallographic methods at limiting resolutions of 1.9, 2.0, and 2.3 Angstrom, respectively. The sulfonamide-zinc binding modes and the association of the ethylene glycol linkers to the hydrophobic patch of the active site are similar in all three inhibitors. Differences in the values of K-d are therefore not due to differences in zinc coordination or to differences in the modes of enzyme-glycol association but instead appear to arise from interaction of the pendant amino acids with the surface of the protein. These pendent groups are, however, not sufficiently ordered to be visible in electron density maps. Thus, structural variations of inhibitors at locations distant from the primary binding (i.e., the sulfonamide group) site affect the overall binding affinities of inhibitors(e.g.; K-d (EG(3)PheNH(3)(+)) = 14 nM as compared with K-d (EG(3)GluNH(3)(+)) = 100 nM).