Cañada F J, Law W C, Rando R R, Yamamoto T, Derguini F, Nakanishi K
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Biochemistry. 1990 Oct 16;29(41):9690-7. doi: 10.1021/bi00493a026.
The biosynthesis of 11-cis-retinol in the retinal pigment epithelium requires two consecutive enzymatic reactions. The first involves the esterification of all-trans-retinol by lecithin retinol acyltransferase (LRAT). The second reaction involves the direct conversion of an all-trans-retinyl ester into 11-cis-retinol by an isomerase-like enzyme. This latter reaction couples the free energy of hydrolysis of an ester to the thermodynamically uphill trans to cis conversion, thus providing the energy to drive the latter process. In this paper both enzymes are studied with respect to their substrate specificities to provide information on mechanism. The isomerase is shown to be highly specific with respect to the ionylidene ring system and substitution at C15, whereas sterically bulkier substituents at C9 and C11 are permitted. C5 and C13 demethyl retinoids are isomerized, removing from consideration isomerization mechanisms involving C-H abstraction at the C5 or C13 methyl groups of the retinoid. On the other hand, C9 demethyl retinoids are not isomerized. A C-H abstraction mechanism is unlikely at the C9 methyl group as well, because no kinetic deuterium isotope effect is found with all-trans-19,19,19-trideuterioretinoids and isomerization of unlabeled retinoids occurs without the incorporation of deuterium when the isomerization is performed in D2O. LRAT proved to be broadly specific for retinols but was relatively inert with other hydrophobic alcohols including cholesterol. The enzyme is also highly specific for phosphatidylcholine analogues versus other potential membranous acyl donors such as phosphatidylethanolamine and phosphatidylserine.
视网膜色素上皮细胞中11-顺式视黄醇的生物合成需要两个连续的酶促反应。第一个反应涉及卵磷脂视黄醇酰基转移酶（LRAT）将全反式视黄醇酯化。第二个反应涉及一种类异构酶将全反式视黄酯直接转化为11-顺式视黄醇。后一个反应将酯水解的自由能与热力学上从反式到顺式的上坡转化耦合，从而提供驱动后一过程的能量。在本文中，对这两种酶的底物特异性进行了研究，以提供有关机制的信息。结果表明，该异构酶对紫罗酮环系统和C15处的取代具有高度特异性，而C9和C11处空间位阻较大的取代基是允许的。C5和C13去甲基类视黄醇发生异构化，排除了涉及类视黄醇C5或C13甲基上C-H提取的异构化机制。另一方面，C9去甲基类视黄醇不发生异构化。C9甲基处也不太可能存在C-H提取机制，因为在全反式-19,19,19-三氘代视黄醇中未发现动力学氘同位素效应，并且当在D2O中进行异构化时，未标记视黄醇的异构化发生时没有氘的掺入。LRAT被证明对视黄醇具有广泛的特异性，但对包括胆固醇在内的其他疏水醇相对不活泼。与其他潜在的膜酰基供体如磷脂酰乙醇胺和磷脂酰丝氨酸相比，该酶对磷脂酰胆碱类似物也具有高度特异性。
