Phosphomannomutase/phosphoglucomutase occupies a central position in the pathways by which several virulence factors are synthesized in Pseudomonas aeruginosa. Virtual screening was used to identify potential inhibitors of phosphomannomutase/ phosphoglucomutase, and one compound, the anthraquinone-based dye Disperse Blue 56, showed potent inhibition in vitro. The kinetics of inhibition was complex; the time courses for reactions in the presence of the inhibitor were biphasic, suggestive of slow-binding inhibition. Quantitative analysis of the progress curves and preincubation experiments demonstrated that slow-binding inhibition was not occurring, however. Initial velocity kinetic studies indicated that Disperse Blue 56 was a parabolic, noncompetitve inhibitor. Progress curves for reactions in the presence of Disperse Blue 56 could be fitted very well by a model in which 2 equiv of the inhibitor bound to free enzyme or the enzyme-substrate complex. The inhibition was largely relieved by the inclusion of 0.01% Triton X-100 in the assay solutions, which has been suggested to be the hallmark for inhibition by compounds that exert their effect through aggregates [McGovern, S. L., Caselli, E., Grigorieff, N., and Shiochet, B. K. (2002) J. Med. Chem. 45, 1712-1722]. Our kinetic data appear to be consistent with either inhibition by a dimer of Disperse Blue 56 or inhibition by a Disperse Blue 56 aggregate, but the latter appears much more likely. We present a detailed analysis of the system to provide further information that may help in the recognition of inhibition through aggregation.