Substrate and stereospecificity of enzymes related to biole acid biosynthesis

生物油酸生物合成相关酶的底物和立体特异性

• 批准号: 11672152
• 负责人: KUROSAWA Takao
• 金额: $ 2.24万
• 依托单位: Health Sciences University of Hokkaido
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11672152/
• 关键词: Bile acid biosynthesis; C27-bile acids; Bile acid CoA ester; HPLC; GC; MS; Peroxisomes; Bifunctional Protein; Sterol Carrier Protein X; ペルオキシゾーム; bifunctional enzyme

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

胆汁酸生物合成中与侧链降解相关的酶（酰基辅酶a氧化酶、两种3-羟基酰基辅酶a水合酶/脱氢酶和酰基辅酶a硫酶）位于肝脏过氧化物酶体中，然而，这些酶的底物特异性和/或立体特异性尚未明确确立。本研究的目的是阐明这些酶的侧链降解的立体化学过程和特异性。首先，我们化学合成了3α、7α、12α-三羟基-5β-胆甾醇-26-油基辅酶a， 3α、7α、12α-三羟基-5β-胆甾醇-24-烯-26-油基辅酶a， 3α、7α、12α、24-四羟基-5β-胆甾醇辅酶a，以及3α、7α、12α-三羟基-24-氧-5β-胆甾醇辅酶a及其类似物，分别作为这三种酶的底物和底物特异性研究。合成了这些辅酶a酯的立体异构体以研究其立体特异性。在此基础上，建立了高效液相色谱法（HPLC）直接分析上述辅酶a酯的定量分析方法。采用高效液相色谱法有效分离了c27 -胆汁酸辅酶a酯的立体异构体。建立了游离c27 -胆汁酸衍生成甲酯-二甲基乙基硅醚后的GC-MS分析方法。这些建立的分析方法对胆汁酸生物合成障碍患者尿液中酶促反应产物和c27 -胆汁酸的分析是非常有效的。利用上述建立的分析方法，对大鼠肝脏过氧化物酶体馏分制备的上述合成辅酶a酯及其相关酶进行产物分析。结果总结如下：在所有酶中均观察到底物特异性。结果表明，末端甲基被3-羟基酰基辅酶a水合酶/脱氢酶（DBP）强烈识别，该酶的水合酶活性为(24R,25R)-3α, 7α, 12α， 24-四羟基-5 - β-胆甾醇辅酶a，其脱氢酶活性进一步脱氢为3α， 7α， 12α-三羟基-24-氧-5 - β-胆甾醇辅酶a。另一种3-羟基酰基辅酶a水合酶/脱氢酶（LBP）可水解出(24S,25S)-3α, 7α, 12α， 24-四羟基-5β-胆固醇酰辅酶a，但LBP不能将其转化为24-氧化合物。这些结果表明，胆汁酸生物合成中的“真正的水合酶/脱氢酶”是DBP。研究了酰基辅酶a硫醇酶的最后降解步骤。甾醇载体蛋白X对24-氧- c27 -胆汁酸具有较强的酰基辅酶a巯基酶活性，是胆汁酸生物合成中的“真巯基酶”。少

项目成果

木村昭彦: "尿中胆汁酸分析による胆汁酸異常症の診断"日本小児科学会雑誌. 104. 686-687 (2000)

Akihiko Kimura：“通过尿胆汁酸分析诊断胆汁酸异常”，日本儿科学会杂志 104. 686-687 (2000)。

Kurosawa T.: "Conjugation reactions catalyzed by bifunctional proteins related to β-oxifdation in bile acid biosynthesis"Steroids. 66. 107-114 (2001)

Kurosawa T.：“胆汁酸生物合成中与 β-氧化相关的双功能蛋白催化的缀合反应”66. 107-114 (2001)

Kurosawa T.: "Conjugation reactions catalyzed by bifunctional proteins related to β-oxifdation in bile acid biosynthesis"Steroids. 66(2). 107-114 (2001)

Kurosawa T.：“胆汁酸生物合成中与 β-氧化相关的双功能蛋白催化的缀合反应”66(2) (2001)。

Kurosawa T.: "Synthesis of Coenzyme A esters of 3α,7α,12α-trihydroxy- and 3α,7α-dihydroxy-24-oxo-5β-cholestan-"Steroids. 66. 499-504 (2001)

Kurosawa T.：“3α，7α，12α-三羟基-和3α，7α-二羟基-24-氧代-5β-胆甾烷-的辅酶A酯的合成”类固醇。 66. 499-504 (2001)

M.BUN-YA, M.MAEBUCHI, S.H.TOGO, T.KUROSAWA, T.HASHIMOTO, T.KAMIRYO: "Metabolic Significance and Expression of Caenorhabditis elegans Tyne II 3-Oxoacyl-CoA Thiolase"Cell Biology and Biophisics. 3. 291-293 (2000)

M.BUN-YA、M.MAEBUCHI、S.H.TOGO、T.KUROSAWA、T.HASHIMOTO、T.KAMIRYO：“秀丽隐杆线虫泰恩 II 3-氧酰辅酶A硫解酶的代谢意义和表达”细胞生物学和生物物理学。