支鏈α-酮酸脫氫酶複合物

支鏈α-酮酸脫氫酶複合物（branched-chain α-ketoacid dehydrogenase complex，BCKDC）是一種在粒線體內膜中找到的酶之多次單元複合物(multi-subunit complex)。^[1] 這種酶複合物催化具支鏈的短鏈α-酮酸的氧化脫羧反應。BCKDC是α-酮酸脫氫酶複合體家族內的成員，包括丙酮酸脫氫酶複合體(pyruvate dehydrogenase)和α-酮戊二酸脫氫酶複合體(α-ketoglutarate dehydrogenase)，是三羧酸循環具有重要功能的酶。

輔助因子

這個複合物的需要下列5個輔因子(cofactor)：

生物上的功能

在動物組織中，BCKDC催化不可逆步驟^[2] ，分解代謝的支鏈氨基酸，即L-異白胺酸，L-纈氨酸和L-白胺酸及其衍生物（分別是L-α-酮-β-甲基戊酸酯，α-酮異戊酸和α-酮異己酸鹽）。^[3]^[4]^[5] 在細菌中，這種酶參與的支鏈、長鏈脂肪酸的合成；^[6]在植物中，這種酶是參與合成支鏈、長鏈的烴。

BCKDC催化的分解代謝之全反應示於圖1。

結構

BCKDC的酶催化機制很大程度上取決於這個大型酶複合體的精細結構。這種酶複合物是由三個催化次單元組成: α-酮酸脫氫酶(alpha-ketoacid dehydrogenase)（E₁部分），二氫硫辛酸轉乙醯基酶(dihydrolipoyl transacylase) （E₂部分），和二氫硫辛醯胺脫氫酶(dihydrolipoamide dehydrogenase)（E₃部分）。在人體中的BCKDC核心中，有24個E₂部分以八面體對稱排列。^[7]24 E₂次單元聚合物分兩部分，12個E₁α2β2四聚體和6個E₃同二聚體以非共價方式結合。除了E₁/E₃-結合區域，在E₂次單元上，有2個其他重要的結構區域：

（i）以該蛋白質的氨基末端部分的硫辛醯-軸承結構域(lipoyl-bearing domain)

（ii）在蛋白質羧基端的內核區域(inner-core domain)。

內核區域是由兩個區間片段（連接子）連接到E₂次單元的其他兩個區域。^[9]內核區域對形成酶複合物的低聚核(oligomeric core)和催化醯基轉移酶的反應是必須的（由“機制”一節中所示）。^[10] E₂的硫辛醯區域可藉由上述提到連結子的彈性構像，使其在組裝好的BCKDC上之E₁、E₂和E₃次單元的活性區位間自由擺動。（參照圖2）^[11]^[12]因此就功能及結構方面來說，E₂部分在BCKDC催化的整個反應扮演著重要的角色。

每個子單元的作用如下

E₁次單元

E₁採用硫胺素焦磷酸（TPP）作為催化的輔助因子。 E₁催化α-酮酸的脫羧反應和後來的硫辛醯結構部分，以共價結合於E₂次單元的還原反應（另一催化輔因子）。

E₂次單元

E₂催化醯基(acyl group)從硫辛醯部分轉至的輔酶A（化學計量的輔因子，stoichiometric cofactor）。^[13]

E₃次單元

在E3₃部分是黃素蛋白(flavoprotein)，且其可作為氧化劑並利用FAD（催化輔因子）重新氧化還原E₂次單元上的硫辛醯硫部分(lipoyl sulfur residues)；然後FAD將這些質子和電子轉移到NAD+（化學計量的輔因子，stoichiometric cofactor）以完成反應循環。

圖5:醯基轉移到輔酶A

圖6:FAD輔酶氧化硫辛醯部分

機制

如前面提到的，在哺乳類動物體內的BCKDC主要功能是，催化支鏈氨基酸分解代謝反應中的不可逆步驟。然而，BCKDC具有相對廣泛的特異性，在比較比例(comparable rates)及對支鏈氨基酸的基質之Km值相似情況下，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)，但這種緩慢速度下，副反應只小具生理意義。^[15]^[16]

其反應機理如下所示。^[17] 請注意，任何一種支鏈 α-酮酸可以作為起始原料；在這個例子中，α-酮異戊酸任意地被選作為BCKDC的基質。

注意：步驟1和2在E₁區域發生。

步驟1: α-酮異戊酸結合TPP，然後進行脫羧反應(decarboxylated)，適當的箭頭推動機構示於圖3。

步驟2：將2-甲基丙醇-TPP(2-methylpropanol-TPP)被氧化形成乙醯基(acetyl group)而被同時轉移到E2中的硫辛酰輔酶。注意，TPP被再生。適當的箭頭推動機構示於圖4。

注：醯化硫辛醯臂(acylated lipoyl arm)現在離開E₁，並盪到E₂的活性區位，在此處發生第3步驟。

步驟3：醯基(Acyl group)轉移到輔酶A(CoA)。適當的箭頭推動機構示於圖5。

注：被還原硫辛醯臂現在盪到在E₃的活性區位，此處步驟4和5發生。

步驟4：將硫辛醯區域被FAD輔酶氧化，如圖所示於圖6。

步驟5： FADH₂再氧化成FAD，產生NADH：NADH::FADH₂ + NAD⁺ --> FAD + NADH + H⁺

疾病相關

缺乏任何這種複酶複合物以或複合物的抑制，會使支鏈氨基酸和它們有害衍生物在體內積聚。這些積累會產生有甜味的排泄物（如耳垢和尿液），及病理學常稱為楓糖尿症。^[18]

在原發性膽汁性肝硬化中，[一種急性肝功能衰竭(acute liver failure)]，這種酶是自身抗原(autoantigen)，這些抗體(antibodies)會辨識氧化的蛋白質，導致炎症免疫反應，有些發炎反應可由麩質過敏解釋。^[19] 其他粒線體自身抗原，可由抗線粒體抗體(anti-mitochondrial antibodies)所辨識的抗原,包括丙酮酸脫氫酶(pyruvate dehydrogenase)和支鏈酮戊二酸脫氫酶(oxoglutarate dehydrogenase)。

參考

^ 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.

外部連結

分类

[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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支鏈α-酮酸脫氫酶複合物

輔助因子

生物上的功能