Kinetic and structural characteristics of the inhibition of enoyl (acyl carrier protein) reductase by triclosan
Kinetic and structural characteristics of the inhibition of enoyl (acyl carrier protein) reductase by triclosan
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DOI：
10.1021/bi9907779
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发表时间：
1999-09-21
期刊：
影响因子：
2.9
通讯作者：
Taylor, IWF
中科院分区：
文献类型：
--
作者：
Ward, WHJ;Holdgate, GA;Taylor, IWF
三氯生被广泛用作皮肤科产品、漱口水和牙膏的抗菌剂。最近的研究表明，抗菌活性是通过与Enoyl(酰基载体蛋白)还原酶(EACPR，EC 1.3.1.9)结合产生的。我们首次在高通量筛选中认识到三氯生抑制EACPR的能力，在高通量筛选中，酶和测试化合物与反应产物NAD(+)预先孵育。抑制50%所需的三氯生浓度接近酶浓度的50%，表明游离化合物通过与EACPR结合而被耗尽。在没有预先孵育或添加NAD(+)的情况下，150 nM三氯生的抑制程度在几分钟内逐渐增加。当加入NAD(+)时，抑制作用出现得更快。凝胶过滤和质谱分析表明，三氯生的抑制作用是可逆的。为了避免游离态抑制剂的耗尽和抑制程度的改变，设计了稳态分析。结果表明，三氯生与E-NAD(+)络合物结合，其解离常数为20~40 pM三氯生对NADH遵循竞争性动力学，在零NADH时的抑制常数为38 pm，并饱和了NAD(+)。在NAD(+)饱和时，NAD(+)的抑制常数为22 pM，表现为非竞争性动力学。通过跟踪三氯生和NAD(+)预孵育的EACPR稀释后的催化活性恢复，测得缓蚀剂的解离速率常数k(OFF)为1.9×10~(-4)S(-1)。结合速率常数(k(On))估计为2.6x10(7)S(-1)M-1。正如预期的那样，k(OFF)/k(ON)=7.1 Pm与稳态研究得到的抑制常数相似。在1.9埃分辨率下测定了大肠杆菌EACPR在辅酶与三氯生的络合物中的晶体结构，表明该化合物与二氮硼类抑制剂的结合部位相似。三氯生的高亲和力似乎是由于在催化中与紧密结合的中间体的结构相似。
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 carrier protein) reductase (EACPR, EC 1.3.1.9). We first recognized the ability of triclosan to inhibit EACPR from Escherichia coli in a high throughput screen where the enzyme and test compound were preincubated with NAD(+), which is a product of the reaction. The concentration of triclosan required for 50% inhibition approximates to 50% of the enzyme concentration, indicating that the free compound is depleted by binding to EACPR. With no preincubation or added NAD(+), the degree of inhibition by 150 nM triclosan increases gradually over several minutes. The onset of inhibition is more rapid when NAD(+) is added. Gel filtration and mass spectrometry show that inhibition by triclosan is reversible. Steady-state assays were designed to avoid depletion of free inhibitor and changes in the degree of inhibition. The results suggest that triclosan binds to E-NAD(+) complex, with a dissociation constant around 20-40 pM. Triclosan follows competitive kinetics with respect to NADH, giving an inhibition constant of 38 pM at zero NADH and saturating NAD(+). Uncompetitive kinetics are observed when NAD(+) is varied, giving an inhibition constant of 22 pM at saturating NAD(+). By following regain of catalytic activity after dilution of EACPR that had been preincubated with triclosan and NAD(+), the 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 started 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 crystal structure of E. coli EACPR in a complex with coenzyme and triclosan has been determined at 1.9 Angstrom resolution, showing that this compound binds in a similar site to the diazaborine inhibitors. The high affinity of triclosan appears to be due to structural similarity to a tightly bound intermediate in catalysis.