Affinity labeling of rat cytochrome P450C24 (CYP24) and identification of Ser57 as an active site residue
Affinity labeling of rat cytochrome P450C24 (CYP24) and identification of Ser57 as an active site residue
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DOI：
10.1016/j.jsbmb.2004.03.107
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发表时间：
2004-05-01
影响因子：
4.1
通讯作者：
Rayb, R
中科院分区：
文献类型：
--
作者：
Omdahl, JL;Swamy, N;Rayb, R
25-羟基维生素D-3或1α，25-二羟基维生素D-3-24R-羟基酶(细胞色素P450C24或细胞色素P450C24)具有清除循环中的25-羟基-D-3和肾脏中过量的1，25(OH)(2)-D-3的双重作用。因此，CYP24是一种重要的多功能调节酶，它维持组织中必需的维生素D激素水平。作为我们对维生素D内分泌系统中各种结合蛋白的结构-功能研究的持续兴趣的一部分，我们用放射性标记的25-OH-D-3亲和类似物来靶向重组大鼠细胞色素P24，并显示该类似物特异性地标记了25-OH-D-3-结合部位。对亲和标记的CYP24样品进行MS/MS分析，确定Ser57是蛋白质全长中唯一被该类似物共价修饰的氨基酸残基。通过定点突变来验证Ser57对底物结合的作用。S57A突变体对25-OH-D-3和1，25(OH)(2)D-3的结合能力显著降低。另一方面，S57D突变体显著增强了底物的结合和1，25(OH)(2)D-3向骨柠檬酸的转化。亲和探针通过25-羟基-D-3的3-羟基锚定。因此，这些结果表明，S57D突变体中的3-羟基(25-OH-D-3和1，25(OH)(2)D-3)可以通过氢键或盐桥稳定下来，从而增强底物亲和力和代谢。(C)2004爱思唯尔有限公司。保留所有权利。
25-hydroxyvitamin D-3- or 1alpha,25-dihydroxyvitamin D-3-24R-hydroxylase (cytochromeP450C24 or CYP24) has a dual role of removing 25-OH-D-3 from circulation and excess 1,25(OH)(2)D-3 from kidney. As a result, CYP24 is an important multifunctional regulatory enzyme that maintains essential tissue-levels of Vitamin D hormone. As a part of our continuing interest in structure-function studies characterizing various binding proteins in the Vitamin D endocrine system, we targeted recombinant rat CYP24 with a radiolabeled 25-OH-D-3 affinity analog, and showed that the 25-OH-D-3-binding site was specifically labeled by this analog. An affinity labeled sample of CYP24 was subjected to MS/MS analysis, which identified Ser57 as the only amino acid residue in the entire length of the protein that was covalently modified by this analog. Site-directed mutagenesis was conducted to validate the role of Ser57 towards substrate-binding. S57A mutant displayed significantly lower binding capacity for 25-OH-D-3 and 1,25(OH)(2)D-3. On the other hand, S57D mutant strongly enhanced binding for the substrates and conversion of 1,25(OH)(2)D-3 to calcitroic acid. The affinity probe was anchored via the 3-hydroxyl group of 25-OH-D-3. Therefore, these results suggested that the 3-hydroxyl group (of 25-OH-D-3 and 1,25(OH)(2)D-3) in the S57D mutant could be stabilized by hydrogen bonding or a salt bridge leading to enhanced substrate affinity and metabolism. (C) 2004 Elsevier Ltd. All rights reserved.