牛肝支链2-氧代酸脱氢酶的部分纯化及性质
Partial purification and properties of branched-chain 2-oxo acid dehydrogenase of ox liver.
作者信息
Parker P J, Randle P J
出版信息
Biochem J. 1978 Jun 1;171(3):751-7. doi: 10.1042/bj1710751.
Abstract
- A branched-chain 2-oxo acid dehydrogenase was partially purified from ox liver mitochondria. 2. The preparation oxidized 4-methyl-2-oxopentanoate, 3-methyl-2-oxobutyrate and D- and L-3-methyl-2-oxopentanoate. The apparent Km values for the oxo acids and for thiamin pyrophosphate, CoA, NAD+ and Mg2+ were determined. 3. The oxidation of each oxo acid was inhibited by isovaleryl (3-methylbutyryl)-CoA (competitive with CoA) and by NADH (competitive with NAD+); Ki values were determined. 4. The preparation showed substrate inhibition with each 2-oxo acid. The oxidative decarboxylation of 4-methyl-2-oxo[1-14C]pentanoate was inhibited by 3-methyl-2-oxobutyrate and DL-3-methyl-2-oxopentanoate, but not by pyruvate. The Vmax. with 3-methyl-2-oxobutyrate as variable substrate was not increased by the presence of each of the other 2-oxo acids. 5. Ox heart pyruvate dehydrogenase did not oxidize these branched-chain 2-oxo acids and it was not inhibited by isovaleryl-CoA. The branched-chain 2-oxo acid dehydrogenase activity (unlike that of pyruvate dehydrogenase) was not inhibited by acetyl-CoA. 6. It is concluded that the branched-chain 2-oxo acid dehydrogenase activity is distinct from that of pyruvate dehydrogenase, and that a single complex may oxidize all three branched-chain 2-oxo acids.
摘要
- 从牛肝线粒体中部分纯化出一种支链2-氧代酸脱氢酶。2. 该制剂可氧化4-甲基-2-氧代戊酸、3-甲基-2-氧代丁酸以及D-和L-3-甲基-2-氧代戊酸。测定了这些氧代酸以及硫胺素焦磷酸、辅酶A、烟酰胺腺嘌呤二核苷酸（NAD⁺）和镁离子（Mg²⁺）的表观米氏常数（Km值）。3. 每种氧代酸的氧化均受到异戊酰（3-甲基丁酰）-辅酶A（与辅酶A竞争性抑制）和NADH（与NAD⁺竞争性抑制）的抑制；测定了抑制常数（Ki值）。4. 该制剂对每种2-氧代酸均表现出底物抑制作用。4-甲基-2-氧代[1-¹⁴C]戊酸的氧化脱羧反应受到3-甲基-2-氧代丁酸和DL-3-甲基-2-氧代戊酸的抑制，但不受丙酮酸的抑制。以3-甲基-2-氧代丁酸为可变底物时，其他2-氧代酸的存在并不会提高其最大反应速度（Vmax）。5. 牛心丙酮酸脱氢酶不能氧化这些支链2-氧代酸，且不受异戊酰辅酶A的抑制。支链2-氧代酸脱氢酶活性（与丙酮酸脱氢酶不同）不受乙酰辅酶A的抑制。6. 得出的结论是，支链2-氧代酸脱氢酶活性与丙酮酸脱氢酶活性不同，且单一复合物可能氧化所有三种支链2-氧代酸。
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