Vessey D A, Hu J, Kelley M
Liver Study Unit, Department of Veteran's Affairs Medical Center, San Francisco, CA 94121, USA.
J Biochem Toxicol. 1996;11(2):73-8. doi: 10.1002/(SICI)1522-7146(1996)11:2<73::AID-JBT4>3.0.CO;2-R.
Neither salicylate nor ibuprofen was a substrate or inhibitor of the long-chain fatty acid:CoA ligase. In contrast, all three xenobiotic-metabolizing medium-chain fatty acid:CoA ligases (XL-I, XL-II, and XL-III) had activity toward salicylate. The K(m) value for salicylate was similar for all three forms (2 to 3 microM), but XL-II and XL-III had higher activity at Vmax. For ibuprofen, only XL-III catalyzed its activation, and it had a K(m) for ibuprofen of 36 microM. Studies of salicylate inhibition of XL-I, XL-II, and XL-III revealed that it inhibited the benzoate activity of all three forms with K1 values of ca. 2 microM, which is in agreement with the K(m) values obtained with salicylate as substrate. Kinetic analysis revealed that salicylate conjugation by all three forms is characterized by substrate inhibition when salicylate exceeds ca. 20 microM. Substrate inhibition was more extensive with XL-I and XL-III. Previous work on the ligases employed assay concentrations of salicylate in the range of 0.1 to 1.0 mM, which are clearly inhibitory, particularly toward XL-I and XL-III. Thus, activity was not properly measured in previous studies, which accounts for the fact that salicylate conjugation was only found with one form, which is most likely XL-II since it has the highest Vmax activity and shows the least amount of substrate inhibition. Studies with ibuprofen indicated that it inhibited XL-I, XL-II, and XL-III, with KI values being in the range of 75-125 microM. The short-chain ligase was inhibited by both salicylate and ibuprofen with KI values of 93 and 84 microM, respectively. It was concluded that pharmacological doses of salicylate, but not ibuprofen, will affect the metabolism of medium-chain fatty acids and carboxylic acid xenobiotics and that the previously described mitochondrial ibuprofen:CoA ligase activity is attributable to XL-III.
水杨酸盐和布洛芬都不是长链脂肪酸辅酶A连接酶的底物或抑制剂。相比之下，所有三种参与外源性物质代谢的中链脂肪酸辅酶A连接酶（XL-I、XL-II和XL-III）对水杨酸盐都有活性。三种形式的酶对水杨酸盐的米氏常数（K(m)）相似（2至3微摩尔），但XL-II和XL-III在最大反应速度（Vmax）时具有更高的活性。对于布洛芬，只有XL-III催化其活化，其对布洛芬的K(m)为36微摩尔。对XL-I、XL-II和XL-III的水杨酸盐抑制研究表明，水杨酸盐抑制这三种形式的苯甲酸活性，抑制常数（K1）值约为2微摩尔，这与以水杨酸盐为底物时获得的K(m)值一致。动力学分析表明，当水杨酸盐超过约20微摩尔时，这三种形式的酶催化水杨酸盐结合的特征是底物抑制。XL-I和XL-III的底物抑制作用更广泛。先前关于连接酶的研究中使用的水杨酸盐测定浓度在0.1至1.0毫摩尔范围内，这显然具有抑制作用，特别是对XL-I和XL-III。因此，先前的研究中没有正确测量活性，这就解释了为什么水杨酸盐结合只在一种形式中被发现，最有可能是XL-II，因为它具有最高的Vmax活性且底物抑制作用最小。对布洛芬的研究表明，它抑制XL-I、XL-II和XL-III，抑制常数（KI）值在75 - 125微摩尔范围内。短链连接酶分别被水杨酸盐和布洛芬抑制，KI值分别为93和84微摩尔。得出的结论是，药理剂量的水杨酸盐而非布洛芬会影响中链脂肪酸和羧酸类外源性物质的代谢，并且先前描述的线粒体布洛芬辅酶A连接酶活性归因于XL-III。
