L-threonine dehydrogenase. Purification and properties of the homogeneous enzyme from Escherichia coli K-12.
L-threonine dehydrogenase. Purification and properties of the homogeneous enzyme from Escherichia coli K-12.
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DOI：
10.1016/s0021-9258(19)69880-7
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发表时间：
1981-02
期刊：
影响因子：
--
通讯作者：
S. Boylan;E. E. Dekker-E.
中科院分区：
文献类型：
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作者：
S. Boylan;E. E. Dekker-E.
L-苏氨酸脱氢酶可能通过α-氨基-β-酮丁酸的中间体形成催化L-苏氨酸转化为氨基丙酮+ CO2，已从大肠杆菌K-12突变体的提取物中纯化至表观均一性，该突变体具有组成性去抑制水平的酶。采用三个分级分离步骤，包括控制热变性、DEAE-Sephadex层析和蓝色葡聚糖-琼脂糖亲和层析。纯化的酶迁移作为一个单一的带，符合脱氢酶活性，当在聚丙烯酰胺凝胶上在pH 8.0和9.5的电泳。在1%十二烷基硫酸钠中电泳也显示出一条带，在沉降速度离心时观察到单一纹影峰。通过蔗糖密度和沉降平衡离心测定，该酶的表观分子量为140，000 +/-4，000。基于在1%十二烷基硫酸钠中的电泳、在6 M盐酸胍中的沉降平衡离心以及与辛二酰亚胺二甲酯的交联，该分子是由相同（或几乎相同）亚基组成的四聚体，其Mr约等于35，000。L-苏氨酸脱氢酶对NAD+或NAD+类似物具有特异性，并利用L-苏氨酸、D-别苏氨酸或L-苏氨酸酰胺作为最佳底物。在50 mM Tris.HCl缓冲液（pH 8.4）和37 ℃下，L-苏氨酸和NAD+的Km值分别为1.43和0.19 mM。该酶的最适pH为10.3，被Mn 2+激活，并且当用某些巯基反应试剂处理时显示出活性的实质性损失。
L-Threonine dehydrogenase, which catalyzes the conversion of L-threonine to aminoacetone + CO2 presumably via the intermediate formation of alpha-amino-beta-ketobutyrate, has been purified to apparent homogeneity from extracts of a mutant of Escherichia coli K-12 which has constitutively derepressed levels of the enzyme. Three fractionation steps were used including controlled heat denaturation, DEAE-Sephadex chromatography, and blue dextran-Sepharose affinity chromatography. The purified enzyme migrated as a single band, coincident with dehydrogenase activity, when electrophoresed on polyacrylamide gels at pH 8.0 and 9.5. Electrophoresis in 1% sodium dodecyl sulfate also showed one band and a single schlieren peak was seen during sedimentation velocity centrifugation. The enzyme has an apparent molecular weight of 140,000 +/- 4,000 as determined by sucrose density and sedimentation equilibrium centrifugation. Based on electrophoresis in 1% sodium dodecyl sulfate, sedimentation equilibrium centrifugation in 6 M guanidine.HCl, and cross-linking with dimethyl suberimidate, the molecule is a tetramer consisting of identical (or nearly identical) subunits with Mr approximately equal to 35,000. L-Threonine dehydrogenase is specific for NAD+ or NAD+ analogs and utilizes L-threonine, D-allothreonine, or L-threonine amide as the best substrates. In 50 mM Tris.HCl buffer (pH 8.4) and 37 degrees C, the Km values for L-threonine and NAD+ are 1.43 and 0.19 mM, respectively. The enzyme has a pH optimum of 10.3, is activated by Mn2+, and shows a substantial loss of activity when treated with certain sulfhydryl-reacting reagents.