Mechanistic and inhibition studies of 4-amino-4-deoxychorismate synthase, anthranilate synthase, isochorismate synthase and salicylate synthase.

摘要

The chorismate-utilizing enzymes 4-amino-4-deoxychorismate synthase (ADCS), isochorismate synthase (IS) and anthranilate synthase (AS) are attractive antimicrobial drug targets. Described is a method to discover compounds that inhibit ADCS. The inhibitor discovery program entails the following: (1) synthesis of a mass-tag encoded library based on a "3-stage" design; (2) massively parallel on-bead screening with fluorescently labeled ADCS; (3) rapid structural identification of hits. The "3-stage" inhibitors of ADCS comprise PAYLOAD and COMBI stages, which interact with active site and surface residues, respectively, and are linked by a SPACER stage. Among eight inhibitors identified by this program, the most potent ADCS inhibitor has a Ki value of 360 microM.;AS, ADCS, IS, and SS each catalyze a nucleophilic substitution reaction, but only IS and SS employ water as a nucleophile. To probe the role of Lys147 as catalytic base, K147Q IS, K147Q SS, Q147K AS, and Q147K ADCS were prepared and enzyme reactions were analyzed by HPLC. The data indicate that Lys147 is not solely responsible for activation of water as a nucleophile. Additional factors that contribute to water activation are proposed.;Among AS, ADCS, IS and SS, only AS and SS possess pyruvate lyase activity. The elimination mechanism was studied by conducting enzyme-catalyzed reactions with protiated chorismate in D2O. 1H NMR analysis of these reaction mixtures indicates that an intramolecular proton transfer does not occur; therefore, AS and SS pyruvate elimination proceeds via an acid/base-assisted mechanism. A change in substrate preference for K147Q SS pyruvate lyase activity indicates Lys147 partially controls SS reaction specificity. Finally, it is demonstrated that AS, ADCS, IS and SS do not possess chorismate mutase promiscuous activity.;The eight compounds identified in the ADCS on-bead screen were subsequently assayed for inhibition of IS and AS. The best overall binding compound inhibits ADCS, IS and AS with Ki values of 720, 56 and 80 microM, respectively. Inhibitors with varying SPACER lengths were tested against ADCS, IS, and AS. Inhibition data confirm the PAYLOAD stage directs the inhibitors to the active site and that substantial binding affinity is added through COMBI---surface residue interactions proximal to the active site.