Kinetic and Structural Characteristics of the Inhibition of Enoyl (Acyl Carrier Protein) Reductase by Triclosan

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• 作者：Walter H. J. Ward ; Geoffrey A. Holdgate ; Siâ ; n Rowsell ; Estelle G. McLean ; Richard A. Pauptit ; Edward Clayton ; Wright W. Nichols ; Jeremy G. Colls ; Claire A. Minshull ; David A. Jude ; Anil Mistry
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：September 21, 1999
• 年：1999
• 卷：38
• 期：38
• 页码：12514 - 12525
• 全文大小：157K
• 年卷期：v.38,no.38(September 21, 1999)
• ISSN：1520-4995

文摘

Triclosan is used widely as an antibacterial agent in dermatological products, mouthwashes,and toothpastes. Recent studies imply that antibacterial activity results from binding to enoyl (acyl carrierprotein) reductase (EACPR, EC 1.3.1.9). We first recognized the ability of triclosan to inhibit EACPRfrom Escherichia coli in a high throughput screen where the enzyme and test compound were preincubatedwith NAD⁺, which is a product of the reaction. The concentration of triclosan required for 50% inhibitionapproximates to 50% of the enzyme concentration, indicating that the free compound is depleted by bindingto EACPR. With no preincubation or added NAD⁺, the degree of inhibition by 150 nM triclosan increasesgradually over several minutes. The onset of inhibition is more rapid when NAD⁺ is added. Gel filtrationand mass spectrometry show that inhibition by triclosan is reversible. Steady-state assays were designedto avoid depletion of free inhibitor and changes in the degree of inhibition. The results suggest that triclosanbinds to E-NAD⁺ complex, with a dissociation constant around 20-40 pM. Triclosan follows competitivekinetics with respect to NADH, giving an inhibition constant of 38 pM at zero NADH and saturatingNAD⁺. Uncompetitive kinetics are observed when NAD⁺ is varied, giving an inhibition constant of 22pM at saturating NAD⁺. By following regain of catalytic activity after dilution of EACPR that had beenpreincubated with triclosan and NAD⁺, the rate constant for dissociation of the inhibitor (k_off) is measuredas 1.9 × 10^-4 s^-1. The association rate constant (k_on) is estimated as 2.6 × 10⁷ s^-1 M^-1 by monitoring theonset of inhibition during assays started by addition of EACPR. As expected, the ratio k_off/k_on = 7.1 pMis similar to the inhibition constants from the steady-state studies. The crystal structure of E. coli EACPRin a complex with coenzyme and triclosan has been determined at 1.9 Å resolution, showing that thiscompound binds in a similar site to the diazaborine inhibitors. The high affinity of triclosan appears to bedue to structural similarity to a tightly bound intermediate in catalysis.