支链α-酮酸脱氢酶复合物

支链α-酮酸脱氢酶复合物（branched-chain α-ketoacid dehydrogenase complex，BCKDC）是一种在线粒体内膜中找到的酶之多次单元复合物(multi-subunit complex)。^[1] 这种酶复合物催化具支链的短链α-酮酸的氧化脱羧反应。BCKDC是α-酮酸脱氢酶复合体家族内的成员，包括丙酮酸脱氢酶复合体(pyruvate dehydrogenase)和α-酮戊二酸脱氢酶复合体(α-ketoglutarate dehydrogenase)，是三羧酸循环具有重要功能的酶。

辅助因子[编辑]

这个复合物的需要下列5个辅因子(cofactor)：

生物上的功能[编辑]

在动物组织中，BCKDC催化不可逆步骤^[2] ，分解代谢的支链氨基酸，即L-异亮氨酸，L-缬氨酸和L-亮氨酸及其衍生物（分别是L-α-酮-β-甲基戊酸酯，α-酮异戊酸和α-酮异己酸盐）。^[3]^[4]^[5] 在细菌中，这种酶参与的支链、长链脂肪酸的合成；^[6]在植物中，这种酶是参与合成支链、长链的烃。

BCKDC催化的分解代谢之全反应示于图1。

结构[编辑]

BCKDC的酶催化机制很大程度上取决于这个大型酶复合体的精细结构。这种酶复合物是由三个催化次单元组成: α-酮酸脱氢酶(alpha-ketoacid dehydrogenase)（E₁部分），二氢硫辛酸转乙酰基酶(dihydrolipoyl transacylase) （E₂部分），和二氢硫辛酰胺脱氢酶(dihydrolipoamide dehydrogenase)（E₃部分）。在人体中的BCKDC核心中，有24个E₂部分以八面体对称排列。^[7]24 E₂次单元聚合物分两部分，12个E₁α2β2四聚体和6个E₃同二聚体以非共价方式结合。除了E₁/E₃-结合区域，在E₂次单元上，有2个其他重要的结构区域：

（i）以该蛋白质的氨基末端部分的硫辛酰-轴承结构域(lipoyl-bearing domain)

（ii）在蛋白质羧基端的内核区域(inner-core domain)。

内核区域是由两个区间片段（连接子）连接到E₂次单元的其他两个区域。^[9]内核区域对形成酶复合物的低聚核(oligomeric core)和催化酰基转移酶的反应是必须的（由“机制”一节中所示）。^[10] E₂的硫辛酰区域可借由上述提到连结子的弹性构像，使其在组装好的BCKDC上之E₁、E₂和E₃次单元的活性区位间自由摆动。（参照图2）^[11]^[12]因此就功能及结构方面来说，E₂部分在BCKDC催化的整个反应扮演着重要的角色。

每个子单元的作用如下

E₁次单元[编辑]

E₁采用硫胺素焦磷酸（TPP）作为催化的辅助因子。 E₁催化α-酮酸的脱羧反应和后来的硫辛酰结构部分，以共价结合于E₂次单元的还原反应（另一催化辅因子）。

E₂次单元[编辑]

E₂催化酰基(acyl group)从硫辛酰部分转至的辅酶A（化学计量的辅因子，stoichiometric cofactor）。^[13]

E₃次单元[编辑]

在E3₃部分是黄素蛋白(flavoprotein)，且其可作为氧化剂并利用FAD（催化辅因子）重新氧化还原E₂次单元上的硫辛酰硫部分(lipoyl sulfur residues)；然后FAD将这些质子和电子转移到NAD+（化学计量的辅因子，stoichiometric cofactor）以完成反应循环。

图5:酰基转移到辅酶A

图6:FAD辅酶氧化硫辛酰部分

机制[编辑]

如前面提到的，在哺乳类动物体内的BCKDC主要功能是，催化支链氨基酸分解代谢反应中的不可逆步骤。然而，BCKDC具有相对广泛的特异性，在比较比例(comparable rates)及对支链氨基酸的底物之Km值相似情况下，也可氧化4-甲硫基-2-氧代丁酸(4-methylthio-2-oxobutyrate)和2-氧代丁酸(2-oxobutyrate)。^[14]BCKDC也可氧化丙酮酸(pyruvate)，但这种缓慢速度下，副反应只小具生理意义。^[15]^[16]

其反应机理如下所示。^[17] 请注意，任何一种支链 α-酮酸可以作为起始原料；在这个例子中，α-酮异戊酸任意地被选作为BCKDC的底物。

注意：步骤1和2在E₁区域发生。

步骤1: α-酮异戊酸结合TPP，然后进行脱羧反应(decarboxylated)，适当的箭头推动机构示于图3。

步骤2：将2-甲基丙醇-TPP(2-methylpropanol-TPP)被氧化形成乙酰基(acetyl group)而被同时转移到E2中的硫辛酰辅酶。注意，TPP被再生。适当的箭头推动机构示于图4。

注：酰化硫辛酰臂(acylated lipoyl arm)现在离开E₁，并荡到E₂的活性区位，在此处发生第3步骤。

步骤3：酰基(Acyl group)转移到辅酶A(CoA)。适当的箭头推动机构示于图5。

注：被还原硫辛酰臂现在荡到在E₃的活性区位，此处步骤4和5发生。

步骤4：将硫辛酰区域被FAD辅酶氧化，如图所示于图6。

步骤5： FADH₂再氧化成FAD，产生NADH：NADH::FADH₂ + NAD⁺ --> FAD + NADH + H⁺

疾病相关[编辑]

缺乏任何这种复酶复合物以或复合物的抑制，会使支链氨基酸和它们有害衍生物在体内积聚。这些积累会产生有甜味的排泄物（如耳垢和尿液），及病理学常称为枫糖尿症。^[18]

在原发性胆汁性肝硬化中，[一种急性肝功能衰竭(acute liver failure)]，这种酶是自身抗原(autoantigen)，这些抗体(antibodies)会辨识氧化的蛋白质，导致炎症免疫反应，有些发炎反应可由麸质过敏解释。^[19] 其他线粒体自身抗原，可由抗线粒体抗体(anti-mitochondrial antibodies)所辨识的抗原,包括丙酮酸脱氢酶(pyruvate dehydrogenase)和支链酮戊二酸脱氢酶(oxoglutarate dehydrogenase)。

参考[编辑]

^ Indo I, Kitano A, Endo F, Akaboshi I, Matsuda, I. Altered Kinetic Properties of the Branched-Chain Alpha-Keto Acid Dehydrogenase Complex Due to Mutation of the Beta-Subunit of the Branched-Chain Alpha-Keto Acid Decarboxylase (E₁) Component in Lymphoblastoid Cells Derived from Patients with Maple Syrup Urine Disease. J Clin Invest. 1987, 80 (1): 63–70. PMID 3597778. doi:10.1172/JCI113064.
^ Yeaman SJ. The 2-oxo acid dehydrogenase complexes: recent advances.. Biochem J. 1989, 257 (3): 625–632. PMID 2649080.
^ Broquist HP, Trupin JS. Amino Acid Metabolism. Annual Review of Biochemistry. 1966, 35: 231–247. doi:10.1146/annurev.bi.35.070166.001311.
^ Harris RA, Paxton R, Powell SM, Goodwin GW, Kuntz MJ, Han AC. Regulation of branched-chain alpha-ketoacid dehydrogenase complex by covalent modification.. Adv Enzyme Regul. 1986, 25: 219–237. PMID 3028049. doi:10.1016/0065-2571(86)90016-6.
^ Namba Y, Yoshizawa K, Ejima A, Hayashi T, Kaneda T. Coenzyme A- and nicotinamide adenine dinucleotide-dependent branched chain alpha-keto acid dehydrogenase. I. Purification and properties of the enzyme from Bacillus subtilis.. J Biol Chem. 1969, 244 (16): 4437–4447. PMID 4308861.
^ Lennarz WJ; et al. The role of isoleucine in the biosynthesis of branched-chain fatty acids by micrococcus lysodeikticus.. Biochemical and Biophysical Research Communications. 1961, 6 (2): 1112–116. PMID 14463994. doi:10.1016/0006-291X(61)90395-3.
^ Aevarsson A, Chuang JL, Wynn RM, Turley S, Chuang DT, Hol WGJ. Crystal structure of human branched-chain α-ketoacid dehydrogenase and the molecular basis of multienzyme complex deficiency in maple syrup urine disease. Structure. 2000, 8 (3): 277–291. PMID 10745006. doi:10.1016/S0969-2126(00)00105-2.
^ Berg, Jeremy M., John L. Tymoczko, Lubert Stryer, and Lubert Stryer. Biochemistry. 6th ed. New York: W.H. Freeman, 2007. 481. Print.
^ Chuang DT. Molecular studies of mammalian branched-chain alpha-keto acid dehydrogenase complexes: domain structures, expression, and inborn errors.. Annals of the New York Academy of Sciences. 1989, 573: 137–154. PMID 2699394. doi:10.1111/j.1749-6632.1989.tb14992.x.
^ Chuang DT, Hu CW, Ku LS, Markovitz PJ, Cox RP. Subunit structure of the dihydrolipoyl transacylase component of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Characterization of the inner transacylase core.. J Biol Chem. 1985, 260 (25): 13779–86. PMID 4055756.
^ Reed LJ, Hackert ML. Structure-function relationships in dihydrolipoamide acyltransferases.. J Biol Chem. 1990, 265 (16): 8971–8974. PMID 2188967.
^ Perham RN. Domains, motifs, and linkers in 2-oxo acid dehydrogenase multienzyme complexes: a paradigm in the design of a multifunctional protein.. Biochemistry. 1991, 30 (35): 8501–8512. PMID 1888719. doi:10.1021/bi00099a001.
^ Heffelfinger SC, Sewell ET, Danner DJ. Identification of specific subunits of highly purified bovine liver branched-chain ketoacid dehydrogenase.. Biochemistry. 1983, 22 (24): 5519–5522. PMID 6652074. doi:10.1021/bi00293a011.
^ Jones SM, Yeaman SJ. Oxidative decarboxylation of 4-methylthio-2-oxobutyrate by branched-chain 2-oxo acid dehydrogenase complex.. Biochemistry. 1986, 237 (2): 621–623. PMC 1147032 . PMID 3800905.
^ Pettit FH, Yeaman SJ, Reed LJ. Purification and characterization of branched chain alpha-keto acid dehydrogenase complex of bovine kidney.. Proceedings of the National Academy of Sciences of the United States of America. 1978, 75 (10): 4881–4885. PMC 336225 . PMID 283398. doi:10.1073/pnas.75.10.4881.
^ Damuni Z, Merryfield ML, Humphreys JS, Reed LJ. Purification and properties of branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney.. Proceedings of the National Academy of Sciences of the United States of America. 1984, 81 (14): 4335–4338. PMC 345583 . PMID 6589597. doi:10.1073/pnas.81.14.4335.
^ Berg, Jeremy M., John L. Tymoczko, Lubert Stryer, and Lubert Stryer. Biochemistry. 6th ed. New York: W.H. Freeman, 2007. 478-79. Print.
^ Podebrad F, Heil M, Reichert S, Mosandl A, Sewell AC, Böhles H. 4,5-dimethyl-3-hydroxy-25H-furanone (sotolone)--the odour of maple syrup urine disease. Journal of Inherited Metabolic Disease. April 1999, 22 (2): 107–114. PMID 10234605. doi:10.1023/A:1005433516026.
^ Leung PS, Rossaro L, Davis PA; et al. Antimitochondrial antibodies in acute liver failure: Implications for primary biliary cirrhosis. Hepatology. 2007, 46 (5): 1436–42. PMID 17657817. doi:10.1002/hep.21828.

外部链接[编辑]

[1] Indo I, Kitano A, Endo F, Akaboshi I, Matsuda, I. Altered Kinetic Properties of the Branched-Chain Alpha-Keto Acid Dehydrogenase Complex Due to Mutation of the Beta-Subunit of the Branched-Chain Alpha-Keto Acid Decarboxylase (E₁) Component in Lymphoblastoid Cells Derived from Patients with Maple Syrup Urine Disease. J Clin Invest. 1987, 80 (1): 63–70. PMID 3597778. doi:10.1172/JCI113064.

[2] Yeaman SJ. The 2-oxo acid dehydrogenase complexes: recent advances.. Biochem J. 1989, 257 (3): 625–632. PMID 2649080.

[3] Broquist HP, Trupin JS. Amino Acid Metabolism. Annual Review of Biochemistry. 1966, 35: 231–247. doi:10.1146/annurev.bi.35.070166.001311.

[4] Harris RA, Paxton R, Powell SM, Goodwin GW, Kuntz MJ, Han AC. Regulation of branched-chain alpha-ketoacid dehydrogenase complex by covalent modification.. Adv Enzyme Regul. 1986, 25: 219–237. PMID 3028049. doi:10.1016/0065-2571(86)90016-6.

[5] Namba Y, Yoshizawa K, Ejima A, Hayashi T, Kaneda T. Coenzyme A- and nicotinamide adenine dinucleotide-dependent branched chain alpha-keto acid dehydrogenase. I. Purification and properties of the enzyme from Bacillus subtilis.. J Biol Chem. 1969, 244 (16): 4437–4447. PMID 4308861.

[6] Lennarz WJ; et al. The role of isoleucine in the biosynthesis of branched-chain fatty acids by micrococcus lysodeikticus.. Biochemical and Biophysical Research Communications. 1961, 6 (2): 1112–116. PMID 14463994. doi:10.1016/0006-291X(61)90395-3.

[7] Aevarsson A, Chuang JL, Wynn RM, Turley S, Chuang DT, Hol WGJ. Crystal structure of human branched-chain α-ketoacid dehydrogenase and the molecular basis of multienzyme complex deficiency in maple syrup urine disease. Structure. 2000, 8 (3): 277–291. PMID 10745006. doi:10.1016/S0969-2126(00)00105-2.

[8] Berg, Jeremy M., John L. Tymoczko, Lubert Stryer, and Lubert Stryer. Biochemistry. 6th ed. New York: W.H. Freeman, 2007. 481. Print.

[9] Chuang DT. Molecular studies of mammalian branched-chain alpha-keto acid dehydrogenase complexes: domain structures, expression, and inborn errors.. Annals of the New York Academy of Sciences. 1989, 573: 137–154. PMID 2699394. doi:10.1111/j.1749-6632.1989.tb14992.x.

[10] Chuang DT, Hu CW, Ku LS, Markovitz PJ, Cox RP. Subunit structure of the dihydrolipoyl transacylase component of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Characterization of the inner transacylase core.. J Biol Chem. 1985, 260 (25): 13779–86. PMID 4055756.

[11] Reed LJ, Hackert ML. Structure-function relationships in dihydrolipoamide acyltransferases.. J Biol Chem. 1990, 265 (16): 8971–8974. PMID 2188967.

[12] Perham RN. Domains, motifs, and linkers in 2-oxo acid dehydrogenase multienzyme complexes: a paradigm in the design of a multifunctional protein.. Biochemistry. 1991, 30 (35): 8501–8512. PMID 1888719. doi:10.1021/bi00099a001.

[13] Heffelfinger SC, Sewell ET, Danner DJ. Identification of specific subunits of highly purified bovine liver branched-chain ketoacid dehydrogenase.. Biochemistry. 1983, 22 (24): 5519–5522. PMID 6652074. doi:10.1021/bi00293a011.

[14] Jones SM, Yeaman SJ. Oxidative decarboxylation of 4-methylthio-2-oxobutyrate by branched-chain 2-oxo acid dehydrogenase complex.. Biochemistry. 1986, 237 (2): 621–623. PMC 1147032 . PMID 3800905.

[15] Pettit FH, Yeaman SJ, Reed LJ. Purification and characterization of branched chain alpha-keto acid dehydrogenase complex of bovine kidney.. Proceedings of the National Academy of Sciences of the United States of America. 1978, 75 (10): 4881–4885. PMC 336225 . PMID 283398. doi:10.1073/pnas.75.10.4881.

[16] Damuni Z, Merryfield ML, Humphreys JS, Reed LJ. Purification and properties of branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney.. Proceedings of the National Academy of Sciences of the United States of America. 1984, 81 (14): 4335–4338. PMC 345583 . PMID 6589597. doi:10.1073/pnas.81.14.4335.

[17] Berg, Jeremy M., John L. Tymoczko, Lubert Stryer, and Lubert Stryer. Biochemistry. 6th ed. New York: W.H. Freeman, 2007. 478-79. Print.

[18] Podebrad F, Heil M, Reichert S, Mosandl A, Sewell AC, Böhles H. 4,5-dimethyl-3-hydroxy-25H-furanone (sotolone)--the odour of maple syrup urine disease. Journal of Inherited Metabolic Disease. April 1999, 22 (2): 107–114. PMID 10234605. doi:10.1023/A:1005433516026.

[pmid17657817-19] Leung PS, Rossaro L, Davis PA; et al. Antimitochondrial antibodies in acute liver failure: Implications for primary biliary cirrhosis. Hepatology. 2007, 46 (5): 1436–42. PMID 17657817. doi:10.1002/hep.21828.

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