Kinetic mechanism of lecithin retinol acyl transferase.
Kinetic mechanism of lecithin retinol acyl transferase.
复制标题
卵磷脂视黄醇酰基转移酶的动力学机制。
DOI：
10.1021/bi00056a009
复制
发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Rando,RR
中科院分区：
文献类型：
--
作者：
Shi,YQ;Hubacek,I;Rando,RR
1992年11月10日接收的摘要：卵磷脂视黄醇酰基转移酶将酰基从卵磷脂的1-位区域特异性地转移到全反式视黄醇（维生素A）和类似的类维生素A。LRAT对于视色素发色团11-ris-retinal的生物合成至关重要，并且对于维生素A的一般饮食动员也是必需的。本文描述了该酶的动力学机制，测定了底物二棕榈酰磷脂酰胆碱（DPPC）的Am和Kmax值[分别为1.38 µ和0.17 µ/（· §）]和全反式-reúnol的Am和Kmax值[分别为0.243 µ和0.199 µ/（· §）]。为了区分乒乓bi-bi机制和快速平衡随机或有序的bi-bi机制，测量了在另一种底物的不同固定浓度下产物形成的速度作为一种底物的函数。产生的平行线完全符合乒乓双双机制，其中DPPC首先结合LRAT和酰化它，并排除简单的随机结合和有序的动力学机制。乒乓双-双机制的进一步证据来自部分交换反应研究，该研究表明LRAT可以催化两种不同卵磷脂衍生物之间的酰基交换。最后，使用有效且可逆的死端抑制剂13-去甲基-13，14-二氢-反式-伊拉黄基三氟乙酸酯，将乒乓反应确立为有序的。证明该化合物相对于DPPC具有竞争性，Ki = 11.4 µ，而相对于全反式/全视黄醇无竞争性。
Revised Manuscript Received November 10, 1992 abstract: Lecithin retinol acyltransferase transfers acyl groups regiospecifically from the 1-position of lecithins to all-trans-retinol (vitamin A) and similar retinoids. LRAT is essential for the biosynthesis of 11-ris-retinal, the visual pigment chromophore, and is also required for the general dietary mobilization of vitamin A. The kinetic mechanism of this enzyme is described here, Am and Kmax values were determined for the substrates dipalmitoylphosphatidylcholine (DPPC)[1.38 µ and 0.17 µ/(· § ), respectively] and for all-trans-reúnol [0.243 µ and 0.199 µ/(· § ), respectively]. In order to distinguish between a ping-pong bi-bi mechanism and a rapid equilibrium random or ordered bi-bi mechanism, the velocity of product formation as a function of one of the substrates at different fixed concentrations of the other substrate was measured. The parallel lines generated are entirely consistent with a ping-pong bi-bi mechanism in which DPPC first binds to LRAT and acylates it and rule out both simple random binding and ordered kinetic mechanisms. Further evidence for a ping-pong bi-bi mechanism comes from partial exchange reaction studies which show that LRAT can catalyze acyl group interchange between two different lecithin derivatives. Finally, the ping-pong reaction was established as being ordered, using the potent and reversible dead-end inhibitor 13-desmethyl-13, 14-dihydro-a//-ira «s-retinyl trifluoroacetate. This compound proved to be competitive with respect to DPPC, with a K\= 11.4 µ, and uncompetitive with respect to all-ira/w-retinol.