Purification and subunit characterization of propanediol dehydratase, a membrane-associated enzyme.
Purification and subunit characterization of propanediol dehydratase, a membrane-associated enzyme.
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丙二醇脱水酶（一种膜相关酶）的纯化和亚基表征。
DOI：
10.1021/bi00518a009
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发表时间：
1981
期刊：
影响因子：
2.9
通讯作者：
Richards,JH
中科院分区：
文献类型：
--
作者：
McGee,DE;Richards,JH
二醇脱氢酶[（J？5）-1，2-丙二醇水解酶; EC4.2. 1.28]是利用腺苷钴胺素（辅酶B12）作为特异性辅因子并催化多种分子重排的一大组酶的实例。在二醇脱氢酶的情况下，反应是1，2-丙二醇重排为1，1-丙二醇，随后立体特异性脱水为丙醛（Zagalak等人，1966; Retey等人，1966年）。腺苷钴胺素辅因子在催化机制中的作用是相当好理解的（Abeles & Zagalak，1966; Frey & Abeles，1966;摩尔等人，1979）;蛋白质在催化中的作用（Bachovchin等人，一种从细菌来源中以纯的形式和高产率分离该酶的有效方法将大大有助于研究该蛋白在催化中的作用，但现有的方法（Abeles，1966; Poznanskaya et al.，1979）没有给出特别高的产率，并且产生的酶似乎在分离过程中经历了显著的蛋白水解。本工作主要研究了一种从肺炎克雷伯氏菌（Klebsiella pneumoniae）中高收率分离纯化丙二醇脱氢酶的方法，并对所得蛋白的亚基结构和60 K亚基的N-末端氨基酸序列进行了初步鉴定。材料和方法细菌。肺炎克雷伯氏菌（ATCC 8724）根据Lee & Abeles（1962）的方法在甘油和1，2-丙二醇存在下生长。生长培养基中使用的自溶啤酒酵母得自Amber Laboratories，Juneau，WI。通过使用配备有JCF-Z转子的Beckman J-21 C连续流离心机收获细菌。称量沉淀的细胞，然后用0.01 M三（羟甲基）氨基甲烷（Tris）1（5 mL/g湿细胞）洗涤，并通过在GSA转子中以10000 rpm离心1 h沉淀。然后将细胞重悬于去离子蒸馏水中并冻干。二醇脱水酶的分离。将冻干的细胞（5-15 g）悬浮于10 mL/g的10 mM DTT和0.1 mM PMSF中，用40%氢氧化钾调节至pH 8.2-8.4。
Diol dehydratase [(J? 5)-1, 2-propanediol hydrolyase; EC4.2. 1.28] from Klebsiella pneumoniae (ATCC 8724) isan example of a large group of enzymes which utilize adeno-sylcobalamin (coenzyme B12) as a specific cofactor and catalyze a variety of molecular rearrangements. In the case of diol dehydratase, the reaction is the rearrangement of 1, 2-propanediol to 1, 1-propanediol and its subsequent stereospecific dehydration to propionaldehyde (Zagalak et al., 1966; Retey et al., 1966). The role of the adenosylcobalamin cofactor in the catalytic mechanism is fairly well understood (Abeles & Zagalak, 1966; Frey & Abeles, 1966; Moore et al., 1979); the role of the protein in catalysis (Bachovchin et al., 1977) re-mains obscure.An efficient procedure for isolating theenzyme in pure form and in high yieldfrom its bacterial source would greatly assist studies of the role of the protein in catalysis, but existing procedures (Abeles, 1966; Poznanskaya et al., 1979) do not give especially high yields and produce enzyme which seems to have undergone significant proteolysis during isolation. This work focuses on a modified procedure for isolating pure propanediol dehydratase from Klebsiella pneumoniae in high yield and the preliminary characterization of the subunit structure of the resulting protein and the N-terminal amino acid se-quence of the 60K subunit. Materials and Methods Bacteria. Klebsiella pneumoniae (ATCC 8724) were grown in thepresence of glycerol and 1, 2-propanediol according to the procedure of Lee & Abeles (1962). Autolyzed brewer’s yeast used in the growth medium was obtained from Amber Laboratories, Juneau, WI. The bacteria were harvested by using a Beckman J-21C continuous-flow centrifuge equipped with a JCF-Z rotor. The pelletedcells were weighed and then washed with 0.01 M tris (hydroxymethyl) aminomethane (Tris) 1 (5 mL/g of wet cells) and pelleted by centrifugation in a GSA rotor for 1 h at 10000rpm. The cells were then resuspended in deionized, distilled water and lyophilized. Isolation of Diol Dehydratase. The lyophilized cells (5-15 g) were suspended in 10 mL/g of 10 mM DTT and 0.1 mM PMSF adjusted to pH 8.2-8.4 with 40% potassium hydroxide.
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DOI：
--
发表时间：
1972
期刊：
Biochimica et Biophysica Acta
影响因子：
--
作者：
Z. Ne'eman;I. Kahane;J. Kovartovsky;S. Razin
通讯作者：
S. Razin
D 和 L 1,2-丙二醇转化为丙醛的立体化学。
影响因子：
4.8
作者：
B. Zagalak;P. Frey;G. Karabatsos;R. Abeles
通讯作者：
R. Abeles
辅酶B12依赖性二醇脱水酶反应中氢转移机制的研究II。
DOI：
--
发表时间：
1971
期刊：
影响因子：
--
作者：
M. Essenberg;P. Frey;R. Abeles
通讯作者：
R. Abeles
膜细胞色素氧化酶的纯化、性质及反应特点
DOI：
--
发表时间：
1966
期刊：
影响因子：
--
作者：
E. Jacobs;E. C. Andrews;W. Cunningham;F. L. Crane
通讯作者：
F. L. Crane
影响因子：
4.8
作者：
R. Abeles;H. A. Lee
通讯作者：
H. A. Lee