Mondal M S, Ruiz A, Bok D, Rando R R
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 45 Shattuck Street, Boston, Massachusetts 02115, USA.
Biochemistry. 2000 May 2;39(17):5215-20. doi: 10.1021/bi9929554.
Lecithin retinol acyltransferase (LRAT) is an essential enzyme in vitamin A metabolism and mobilization. The membrane-bound enzyme catalyzes the transfer of an acyl group from the sn-1 position of lecithin to vitamin A to generate retinyl esters. The sequence of LRAT is novel and hence does not suggest a mechanistic class to which the enzyme belongs. However, the activity of the enzyme is exceedingly sensitive to affinity labeling and group-specific reagents directed toward thiol groups. LRAT from human retinal pigment epithelium has cysteine residues at positions 161, 168, 182, and 208. Site-specific mutagenic studies show that C182 and C208 can be converted to alanines with little affect on activity. The activities of the C161A and C168A mutants are virtually nil. Moreover, while C168S is substantially active, C161S possesses only a few percent of the activity of wild-type (WT) LRAT. Also, pH-rate profiles show that C168S has virtually the same profile as WT LRAT, while C161S shows an aberrant profile quite unlike that of WT LRAT. Therefore, LRAT is a thiol acyltransferase and C161 may be the essential nucleophilic residue critical for catalysis.
卵磷脂视黄醇酰基转移酶（LRAT）是维生素A代谢和转运过程中的一种关键酶。这种膜结合酶催化卵磷脂sn-1位上的酰基转移至维生素A，生成视黄酯。LRAT的序列是全新的，因此无法表明该酶所属的作用机制类别。然而，该酶的活性对亲和标记和针对巯基的基团特异性试剂极为敏感。来自人视网膜色素上皮的LRAT在第161、168、182和208位含有半胱氨酸残基。定点诱变研究表明，C182和C208突变为丙氨酸对活性影响很小。C161A和C168A突变体的活性几乎为零。此外，虽然C168S具有较高活性，但C161S仅具有野生型（WT）LRAT百分之几的活性。而且，pH-速率曲线表明，C168S与WT LRAT的曲线几乎相同，而C161S则呈现出与WT LRAT截然不同的异常曲线。因此，LRAT是一种巯基酰基转移酶，C161可能是催化作用中至关重要的亲核残基。
