Substrate and stereospecificity of enzymes related to biole acid biosynthesis

胆汁酸生物合成中与侧链降解有关的酶(酰基辅酶A氧化酶、两种3-羟基辅酶A水合/脱氢酶和酰基辅酶A硫解酶)位于肝脏过氧化物体中，但这些酶的底物特异性和/或立体特异性尚未明确。首先，我们化学合成了3-α、7-α、12-α-三羟基-5-β-胆甾烷-26-甲酰辅酶A、3-α、7-α、12-α-三羟基-5-β-胆甾醇-24-烯-26-甲酰辅酶A、3-α、7-α、12-α、24-四羟基-5-β-胆烷基辅酶A和3-α、7-α、12-α-三羟基-24-氧代-5-β-胆烷基辅酶A及其类似物作为这三种酶的底物。我们还合成了这些辅酶A酯的立体异构体，用于立体专一性的研究。然后，我们建立了直接测定t-…的高效液相色谱定量分析方法比可可酯更多的是他。用高效液相色谱法对C27-胆汁酸辅酶A酯的立体异构体进行了有效分离。并建立了GC-MS法分析衍生化为甲酯-二甲基乙基硅醚后的游离C27-胆汁酸。建立的分析方法对胆汁酸生物合成障碍患者尿液中胆汁酸的酶反应产物和C27-胆汁酸的分析是高效的。实验结果总结如下：在所有的酶中观察到底物的特异性。3-羟基酰辅酶A水合脱氢酶(3-羟基酰辅酶A水合/脱氢酶)对末端甲基有较强的识别作用，该酶通过水合作用给出(24R，25R)-3α，7α，12α，24-四羟基-5-β-胆烷酰辅酶A的水合活性，而脱氢酶活性被进一步脱氢生成3-α，7-α，12-α-三羟基-24-氧代-5-β-胆烷基辅酶A。另一种3-羟基酰辅酶A水合/脱氢酶不能将其转化为24-氧-化合物。这些结果表明胆汁酸生物合成中真正的水合/脱氢酶是DBP，最后一步是酰基辅酶A硫解酶的降解。研究发现，Sterol载体蛋白X对24-氧代-C27-胆汁酸具有很强的酰基辅酶活性，表明胆汁酸的生物合成是“真正的硫解酶”。较少

The enzymes (acyl-CoA oxidase, two 3-hydroxyacyl-CoA hydratase/dehydrogenase and acyl-CoA thiolase) related to side chain degradation in bile acid biosynthesis are located in liver peroxisomes, however, the substrate specificities and/or stereospecificiies of these enzymes have not been clearly established. The aime of the present study is to clarify the stereochemical course of the side chain degradation and specificities of these enzymes.First, we chemically synthesized 3α, 7α, 12α-trihydroxy-5β-cholestan-26-oyl CoA, 3α, 7α, 12α-trihydroxy-5β-cholest-24-en-26-oyl CoA, 3α, 7α, 12α, 24-tetrahydroxy-5β-cholestanoyl CoA, and 3α, 7α, 12α-trihydroxy-24-oxo-5β-cholestanoyl CoA as the substrates for these three enzymes, respectively, and analogues of these CoA esters for the study of substrate specificities. The stereoisomers of these CoA esters were also synthesized for the study of stereospecificities.Then, we developed the quantitative analytical method using HPLC for direct analysis of t … More he above CoA esters . The stereoisomers of C27-bile acid CoA esters were effectively separated by HPLC method. And also GC-MS method was also established for the analysis of free C27-bile acids after derivatization into methyl ester-di-methylethylsilyl ethers. These established analytical methods were highly effective for the analysis of products of enzymatic reactions and C27-bile acids in urine of patients with bile acid biosynthesis disorder.The products analysis of the above synthesized CoA esters with related enzymes, which were prepared from rat liver peroxisomal fractions, were carried out using above established analytical method. The results are summarized as follows ;The substrate specificities were observed in all enzymes. It was noted that the terminal methyl group is strongly recognized by 3-hydroxyacyl-CoA hydratase/dehydrogenase (DBP), and also this enzyme give (24R,25R)-3α, 7α, 12α, 24-tetrahydroxy-5β-cholestanoyl CoA as its hydratase activity, which is further dehydroganated its dehydrogenase activity to give 3α, 7α, 12α-trihydroxy-24-oxo-5β-cholestanoyl CoA.Another 3-hydroxyacyl-CoA hydratase/dehydrogenase (LBP) gave (24S,25S)-3α, 7α, 12α, 24-tetrahydroxy-5β-cholestanoyl CoA by its hydratse, however, LBP could not convert it to 24-oxo-compound. These results indicated that the "real e hydratse/dehydrogenase" in bile acid biosynthesis is DBP.The last degradation step by acyl-CoA thiolase was also investigated. It was found that Sterol carrier protein X has strong acyl CoA tholase activity for 24-oxo-C27-bile acids indicating the "true thiolase" in biosynthesis of bile acid. Less