Kinetic and structural characteristics of the inhibition of enoyl (acyl carrier protein) reductase by triclosan

Triclosan is used widely as an antibacterial agent in dermatological produc ts, mouthwashes, and toothpastes. Recent studies imply that antibacterial a ctivity results from binding to enoyl (acyl carrier protein) reductase (EAC PR, EC 1.3.1.9). We first recognized the ability of triclosan to inhibit EA CPR from Escherichia coli in a high throughput screen where the enzyme and test compound were preincubated with NAD(+), which is a product of the reac tion. The concentration of triclosan required for 50% inhibition approximat es to 50% of the enzyme concentration, indicating that the free compound is depleted by binding to EACPR. With no preincubation or added NAD(+), the d egree of inhibition by 150 nM triclosan increases gradually over several mi nutes. The onset of inhibition is more rapid when NAD(+) is added. Gel filt ration and mass spectrometry show that inhibition by triclosan is reversibl e. Steady-state assays were designed to avoid depletion of free inhibitor a nd changes in the degree of inhibition. The results suggest that triclosan binds to E-NAD(+) complex, with a dissociation constant around 20-40 pM. Tr iclosan follows competitive kinetics with respect to NADH, giving an inhibi tion constant of 38 pM at zero NADH and saturating NAD(+). Uncompetitive ki netics are observed when NAD(+) is varied, giving an inhibition constant of 22 pM at saturating NAD(+). By following regain of catalytic activity afte r dilution of EACPR that had been preincubated with triclosan and NAD(+), t he rate constant for dissociation of the inhibitor (k(off)) is measured as 1.9 x 10(-4) s(-1). The association rate constant (k(on)) is estimated as 2 .6 x 10(7) s(-1) M-1 by monitoring the onset of inhibition during assays st arted by addition of EACPR. As expected, the ratio k(off)/k(on) = 7.1 pM is similar to the inhibition constants from the steady-state studies. The cry stal structure of E. coli EACPR in a complex with coenzyme and triclosan ha s been determined at 1.9 Angstrom resolution, showing that this compound bi nds in a similar site to the diazaborine inhibitors. The high affinity of t riclosan appears to be due to structural similarity to a tightly bound inte rmediate in catalysis.