School of Chemistry and Biochemistry, The University of Western Australia, Crawley, Australia.
FEBS J. 2014 Jul;281(14):3280-96. doi: 10.1111/febs.12862. Epub 2014 Jun 25.
CYP24A1 is the multicatalytic cytochrome P450 responsible for the catabolism of vitamin D via the C23- and C24-oxidation pathways. We successfully expressed the labile human enzyme in Escherichia coli and partially purified it in an active state that permitted detailed characterization of its metabolism of 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3] and the intermediates of the C24-oxidation pathway in a phospholipid-vesicle reconstituted system. The C24-oxidation pathway intermediates, 1,24,25-trihydroxyvitamin D3, 24-oxo-1,25-dihydroxyvitamin D3, 24-oxo-1,23,25-trihydroxyvitamin D3 and tetranor-1,23-dihydroxyvitamin D3, were enzymatically produced from 1,25(OH)2 D3 using rat CYP24A1. Both 1,25(OH)2 D3 and 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 were found to partition strongly into the phospholipid bilayer when in aqueous medium. Changes to the phospholipid concentration did not affect the kinetic parameters for the metabolism of 1,25(OH)2 D3 by CYP24A1, indicating that it is the concentration of substrates in the membrane phase (mol substrate·mol phospholipid(-1) ) that determines their rate of metabolism. CYP24A1 exhibited Km values for the different C24-intermediates ranging from 0.34 to 15 mmol·mol phospholipid(-1) , with 24-oxo-1,23,25-trihydroxyvitamin D3 [24-oxo-1,23,25(OH)3 D3] displaying the lowest and 1,24,25-trihydroxyvitamin D3 [1,24,25(OH)3 D3] displaying the highest. The kcat values varied by up to 3.8-fold, with 1,24,25(OH)3 D3 displaying the highest kcat (34 min(-1) ) and 24-oxo-1,23,25(OH)3 D3 the lowest. The data show that the cleavage of the side chain of 24-oxo-1,23,25(OH)3 D3 occurs with the highest catalytic efficiency (kcat /Km ) and produces 1-hydroxy-23-oxo-24,25,26,27-tetranorvitamin D3 and not 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3, as the primary product. These kinetic analyses also show that intermediates of the C24-oxidation pathway effectively compete with precursor substrates for binding to the active site of the enzyme, which manifests as an accumulation of intermediates, indicating that they dissociate after each catalytic step.
CYP24A1 是一种多催化细胞色素 P450，负责通过 C23-和 C24-氧化途径代谢维生素 D。我们成功地在大肠杆菌中表达了不稳定的人酶，并在活性状态下部分纯化了它，这使得我们能够在磷脂囊泡重建系统中详细表征其代谢 1,25-二羟维生素 D3[1,25(OH)2D3]和 C24-氧化途径的中间产物。C24-氧化途径中间产物，1,24,25-三羟维生素 D3、24-氧-1,25-二羟维生素 D3、24-氧-1,23,25-三羟维生素 D3 和四氢诺-1,23-二羟维生素 D3，使用大鼠 CYP24A1 从 1,25(OH)2D3 酶促生成。1,25(OH)2D3 和 1,23-二羟-24,25,26,27-四氢诺维生素 D3 均被发现当处于水介质中时强烈分配到磷脂双层中。磷脂浓度的变化并不影响 CYP24A1 代谢 1,25(OH)2D3 的动力学参数，这表明是底物在膜相中的浓度（mol 底物·mol 磷脂(-1)）决定了它们的代谢速率。CYP24A1 对不同 C24 中间产物的 Km 值范围为 0.34 至 15mmol·mol 磷脂(-1)，其中 24-氧-1,23,25-三羟维生素 D3[24-氧-1,23,25(OH)3D3]的 Km 值最低，而 1,24,25-三羟维生素 D3[1,24,25(OH)3D3]的 Km 值最高。kcat 值变化高达 3.8 倍，其中 1,24,25(OH)3D3 的 kcat 值最高（34min(-1)），而 24-氧-1,23,25(OH)3D3 的 kcat 值最低。数据表明，24-氧-1,23,25(OH)3D3 侧链的裂解具有最高的催化效率（kcat/Km），并产生 1-羟基-23-氧-24,25,26,27-四氢诺维生素 D3，而不是 1,23-二羟-24,25,26,27-四氢诺维生素 D3，作为主要产物。这些动力学分析还表明，C24-氧化途径的中间产物有效地与前体底物竞争与酶的活性位点结合，这表现为中间产物的积累，表明它们在每个催化步骤后解离。
