25-Hydroxyvitamin D3 3-epimerase catalyzes the 3β → 3α epimerization of 25-hydroxyvitamin D3 (25(OH)D3) producing 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3). 3-Epi-25(OH)D3 is one of the most abundant forms of vitamin D present in the serum. It can be converted to 3-epi-1α,25-dihydroxyvitamin D3 by CYP27B1 which generally displays lower biological activity than 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). The 25(OH)D3 3-epimerase has been poorly characterized to date and the gene encoding it has not been identified. The 3-epimerase has been reported to be present in the microsomal fraction of cells, including liver cells, and to use NADPH as cofactor. It can also act on 1,25(OH)2D3 and 24,25(OH)2D3 forming the 3α-epimers. In this study we have characterized the activity of the 25(OH)D3 3-epimerase in rat and human liver microsomes, using 25(OH)D3 as substrate and HPLC to analyze product formation. For both rat and human liver microsomes the preferred cofactor was NADH, with the rat enzyme displaying a 6-fold greater catalytic efficiency (Vmax/Km) for NADH over that for NADPH. No activity was observed with oxidized cofactor, either NAD+ or NADP+. This was unexpected since the initial step in the epimerization, predicted to be the oxidation of the 3β-OH to a ketone, would require oxidized cofactor. The rat 3-epimerase in microsomes gave a Km for 25(OH)D3 of 14 μM. The reverse reaction, conversion of 3-epi-25(OH)D3 to 25(OH)D3, was catalyzed by both rat and human liver microsomes but at lower rates than the forward reaction. In conclusion, both rat and human 25-hydroxyvitamin D3 3-epimerase catalyze the reversible interconversion of 25(OH)D3 and 3-epi-25(OH)D3, and use NADH as the preferred cofactor. The lack of requirement for exogenous NAD+ suggests that the enzyme has a tightly bound NAD+ in its active site that is released only upon its reduction.

中文翻译：

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25-羟基维生素D3 3-表异构酶在大鼠和人肝微粒体中的催化特性

25-羟基维生素D3 3-表异构酶催化25-羟基维生素D3（25（OH）D3）的3β→3α差向异构化反应，生成3-epi-25-羟基维生素D3（3-epi-25（OH）D3）。3-Epi-25（OH）D3是血清中存在的最丰富的维生素D形式之一。CYP27B1可以将其转化为3-epi-1α，25-二羟基维生素D3，其通常具有比1α，25-二羟基维生素D3（1,25（OH）2D3）更低的生物活性。迄今为止，25（OH）D3 3-表异构酶的表征较差，尚未鉴定出编码它的基因。据报道，3-表异构酶存在于包括肝细胞在内的细胞微粒体中，并使用NADPH作为辅因子。它也可以作用于1,25（OH）2D3和24,25（OH）2D3，形成3α-受体。在这项研究中，我们表征了大鼠和人类肝脏微粒体中25（OH）D3 3-表异构酶的活性，使用25（OH）D3作为底物并用HPLC分析产物形成。对于大鼠和人肝微粒体，优选的辅因子是NADH，其中大鼠酶对NADH的催化效率（Vmax / Km）比对NADPH的催化效率高6倍。用氧化的辅因子NAD +或NADP +没有观察到活性。这是出乎意料的，因为在差向异构化的初始步骤（预计是3β-OH氧化为酮）将需要氧化的辅因子。微粒体内的大鼠3-表异构酶的25（OH）D3的Km为14μM。大鼠和人的肝微粒体均催化逆反应，即3-epi-25（OH）D3转化为25（OH）D3，但速率低于正反应。总之，大鼠和人的25-羟基维生素D3 3-表异构酶均催化25（OH）D3和3-epi-25（OH）D3的可逆相互转化，并使用NADH作为首选辅因子。对外源NAD +的缺乏需求表明该酶在其活性位点具有紧密结合的NAD +，该酶仅在还原时才释放。