STRUCTURE OF 3,4-DICHLOROISOCOUMARIN-INHIBITED FACTOR-D

Factor D (D) is a serine protease essential in the activation of the a lternative complement pathway. Only a few of the common serine proteas e inhibitors inhibit D, binding covalently to the serine hydroxyl of t he catalytic triad. 3,4-Dichloroisocoumarin (DCI) is a mechanism-based inhibitor which inhibits most serine proteases and many esterases, in cluding D. The structure of the enzyme:inhibitor covalent adduct of D with DCI, DCI:D, to a resolution of 1.8 Angstrom is described, which r epresents the first structural analysis of D with a mechanism-based in hibitor. The side chain of the ring-opened DCI moiety of the protein a dduct undergoes chemical modification in the buffered solution, result ing in the formation of an cr-hydroxy acid moiety through the nucleoph ilic substitution of both Cl atoms. The inhibited enzyme is similar in overall structure to the native enzyme, as well as to a variety of is ocoumarin-inhibited trypsin and porcine pancreatic elastase (PPE) stru ctures, yet notable differences are observed in the active site and bi nding mode of these small-molecule inhibitors. One region of the activ e site (residues 189-195) is relatively conserved between factor D, tr ypsin, and elastase with respect to amino-acid sequence and to conform ation. Another region (residues 214-220) reflects the amino-acid subst itutions and conformational flexibility between these enzymes. The car bonyl O atom of the DCI moiety was found to be oriented away from the oxyanion hole, which greatly contributes to the stability of the DCI:D adduct. The comparisons of the active sites between native factor D, DCI-inhibited factor D, and various inhibited trypsin and elastase (PP E) molecules are providing the chemical bases directing the design of novel, small-molecule pharmaceutical agents capable of modulating the alternative complement pathway.