Diczfalusy M A, Björkhem I, Einarsson C, Alexson S E
Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, Sweden.
Eur J Biochem. 1999 Jan;259(1-2):404-11. doi: 10.1046/j.1432-1327.1999.00052.x.
Fatty acid ethyl esters have been detected in high concentrations in organs commonly damaged by alcohol abuse and are regarded as being important non-oxidative metabolites of ethanol. The formation of fatty acid ethyl esters (FAEEs) has been ascribed to two enzymic activities, acyl-CoA : ethanol O-acyltransferase (AEAT) and FAEE synthase. In the present study we determined AEAT and FAEE synthase activities in isolated rat liver microsomes and further characterized the microsomal AEAT activity in more detail. The determined AEAT and FAEE synthase activities were found to be similar (about 1.7 nmol.min-1.mg-1). However, the AEAT activity was increased about sixfold by the addition of 250 microm bis-(4-nitrophenyl) phosphate (a serine esterase inhibitor) to the incubation whereas FAEE synthase activity was completely inhibited. p-Hydroxymercuribenzoic acid (a cysteine-reacting compound) also stimulated AEAT activity (about fourfold) but had no effect on FAEE synthase activity. The effects of the inhibitors suggest that the formation of FAEEs by AEAT was severely counteracted by enzymic hydrolysis of the substrate (acyl-CoA) and to a lesser extent the product by serine esterases. dl-Melinamide, a hypocholesterolaemic drug, was found to be a very potent inhibitor of AEAT activity with an IC50 value of about 2.5 microm. Furthermore, we compared the activities of two purified microsomal carboxylesterases, ES-4 and ES-10, and identified ES-4 as the enzyme responsible for hydrolysis of FAEEs. The two carboxyesterases were also tested for FAEE synthase activity, but neither had any detectable activity. Esterase ES-4 was found to have some AEAT activity, but it was low. When measured under optimal conditions without competing hydrolysis the capacity of AEAT is thus considerably higher than FAEE synthase and the results are consistent with an important role for AEAT in the formation of ethyl esters. As the ratio acyl-CoA/non-esterified fatty acids is high under normal conditions, AEAT is probably the most important enzyme in fatty acid ethyl ester formation.
在通常因酒精滥用而受损的器官中已检测到高浓度的脂肪酸乙酯，并且它们被视为乙醇重要的非氧化代谢产物。脂肪酸乙酯（FAEEs）的形成归因于两种酶活性，即酰基辅酶A：乙醇O-酰基转移酶（AEAT）和FAEE合酶。在本研究中，我们测定了分离的大鼠肝微粒体中的AEAT和FAEE合酶活性，并进一步更详细地表征了微粒体AEAT活性。所测定的AEAT和FAEE合酶活性相似（约1.7 nmol·min⁻¹·mg⁻¹）。然而，在孵育体系中加入250微摩尔双（4-硝基苯基）磷酸酯（一种丝氨酸酯酶抑制剂）后，AEAT活性增加了约6倍，而FAEE合酶活性则被完全抑制。对羟基汞苯甲酸（一种与半胱氨酸反应的化合物）也刺激了AEAT活性（约4倍），但对FAEE合酶活性没有影响。这些抑制剂的作用表明，AEAT形成FAEEs的过程受到底物（酰基辅酶A）的酶促水解以及丝氨酸酯酶对产物的水解的严重抵消，程度较轻。降血脂药物dl-美拉酰胺被发现是AEAT活性的一种非常有效的抑制剂，IC50值约为2.5微摩尔。此外，我们比较了两种纯化的微粒体羧酸酯酶ES-4和ES-10的活性，并确定ES-4是负责水解FAEEs的酶。还测试了这两种羧酸酯酶的FAEE合酶活性，但两者均无任何可检测到的活性。发现酯酶ES-4具有一些AEAT活性，但很低。因此，在没有竞争性水解的最佳条件下测量时，AEAT的能力明显高于FAEE合酶，结果与AEAT在乙酯形成中的重要作用一致。由于在正常条件下酰基辅酶A/非酯化脂肪酸的比例很高，AEAT可能是脂肪酸乙酯形成中最重要的酶。
