Dilworth F J, Scott I, Green A, Strugnell S, Guo Y D, Roberts E A, Kremer R, Calverley M J, Makin H L, Jones G
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
J Biol Chem. 1995 Jul 14;270(28):16766-74. doi: 10.1074/jbc.270.28.16766.
A series of homologated 1 alpha-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 molecules with one to three extra carbons in the side chain were used to examine the substrate preferences and hydroxylation site selection mechanisms of the liver vitamin D3-25-hydroxylase (CYP27) and the target cell 25-hydroxyvitamin D3-24-hydroxylase (CYP24). Cultured and transfected cell models, used as sources of these hydroxylases, gave 23-, 24-, 25-, and 27-hydroxylated metabolites which were identified by their high performance liquid chromatography and GC-MS characteristics. Lengthening the side chain is tolerated by each cytochrome P450 isoform such that 25-hydroxylation or 24-hydroxylation continues to occur at the same rate as in the native side chain, while the site of hydroxylation remains the same for the liver enzyme in that CYP27 continues to hydroxylate at C-25 and C-27 (minor) despite the two-carbon-atom extension. Somewhat surprising is the finding that C-24 and C-23 (minor) hydroxylations also do not change as the side chain is extended by as much as three carbons. We conclude that CYP24 must be directed to its hydroxylation site(s) by the distance of carbon 24 from the vitamin D ring structure and not as in CYP27 by the distance of the hydroxylation site from the end of the side chain.
一系列侧链带有1至3个额外碳原子的同系化1α-羟基维生素D3和1,25-二羟基维生素D3分子被用于研究肝脏维生素D3-25-羟化酶（CYP27）和靶细胞25-羟基维生素D3-24-羟化酶（CYP24）的底物偏好和羟基化位点选择机制。用作这些羟化酶来源的培养和转染细胞模型产生了23-、24-、25-和27-羟基化代谢产物，这些代谢产物通过其高效液相色谱和气相色谱-质谱特征得以鉴定。每种细胞色素P450同工型都能耐受侧链的延长，使得25-羟基化或24-羟基化继续以与天然侧链相同的速率发生，而对于肝脏酶来说，羟基化位点保持不变，即尽管侧链延长了两个碳原子，CYP27仍继续在C-25和C-27（次要）处进行羟基化。有点令人惊讶的是，发现随着侧链延长多达三个碳原子，C-24和C-23（次要）羟基化也没有变化。我们得出结论，CYP24必须通过碳24与维生素D环结构的距离被引导至其羟基化位点，而不像CYP27那样通过羟基化位点与侧链末端的距离来引导。
