Purification and substrate characterization of α-ketobutyrate decarboxylase from Pseudomonas putida
Purification and substrate characterization of α-ketobutyrate decarboxylase from Pseudomonas putida
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DOI：
10.1016/s1381-1177(03)00089-4
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发表时间：
2003-09-01
影响因子：
--
通讯作者：
Tanaka, H
中科院分区：
文献类型：
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作者：
Inoue, H;Nishito, A;Tanaka, H
恶臭假单胞菌L-甲硫氨酸催化剂操纵子中编码的α-酮丁酸脱羧酶与革兰氏阴性菌丙酮酸脱氢酶复合物的E1组分同源。该酶从大肠杆菌的细胞提取物中纯化至均一。纯化的酶在SDS-PAGE上为同源二聚体，亚基M-r为93，000。通过添加焦磷酸硫胺素（TPP）和二价阳离子如Mg 2+、Mn 2+和Co 2+来激活酶活性。该酶对α-酮丁酸和α-酮-正戊酸而不是丙酮酸显示出高活性，但具有增加的侧链长度以及分支的α-酮酸，如α-酮-正己酸和α-酮-3-甲基戊酸，不被该酶使用。α-酮丁酸和丙酮酸的Km值分别为0.016和0.147 mM，α-酮丁酸的k（cat）/Km值（10.69 s（-1）mM（-1））是丙酮酸的29倍。因此，α-酮丁酸脱羧酶与丙酮酸脱氢酶E1组分在底物特异性方面不同，尽管它们的结构和酶学性质相似。这些结果表明，α-酮丁酸脱羧酶独特的底物特异性是由于两种酶的高度保守的活性位点略有不同。(C)2003 Elsevier B. V.保留所有权利。
alpha-Ketobutyrate decarboxylase encoded in the L-methionine catabolism operon of Pseudomonas putida is homologous with the E1 component of pyruvate dehydrogenase complex from gram-negative bacteria. The enzyme was purified to homogeneity from the cell extract of an Escherichia coli transformant. The purified enzyme was homodimeric with a subunit of M-r 93,000 on SDS-PAGE. The enzyme activity was activated by the addition of both thiamine pyrophosphate (TPP) and a divalent cation, such as Mg2+ Mn2+, and Co2+. The enzyme showed high activity for alpha-ketobutyrate and alpha-keto-n-valerate rather than pyruvate, but the alpha-keto acids with increasing length of the side chain as well as branching, such as alpha-keto-n-caproate and alpha-keto-3-methylvalerate, were not used by the enzyme. The K-m values for alpha-ketobutyrate and pyruvate were 0.016 and 0.147 mM, respectively, and the k(cat)/K-m value(10.69 s(-1) mM(-1))for alpha-ketobutyrate was 29-fold greater than that for pyruvate. Thus, alpha-ketobutyrate decarboxylase is distinguished from the pyruvate dehydrogenase E1 component with respect to the substrate specificity, although their structural and enzymological properties were similar. These results suggest that the unique substrate specificity of alpha-ketobutyrate decarboxylase is due to a slight difference in the highly conserved active sites of both enzymes. (C) 2003 Elsevier B.V. All rights reserved.