Hanschmann H, Kleber H P
Institut für Biochemie, Fakultät für Biowissenschaften, Pharmazie und Psychologie, Universität Leipzig, Germany.
Biochim Biophys Acta. 1997 Jan 4;1337(1):133-42. doi: 10.1016/s0167-4838(96)00161-6.
D(+)-Carnitine dehydrogenase from Agrobacterium sp. catalyzes the oxidation of D(+)-carnitine to 3-dehydrocarnitine as initial step of D(+)-carnitine degradation. The NAD(+)-specific, cytosolic enzyme was purified 126-fold to apparent electrophoretic homogeneity by 4 chromatographic steps. The molecular mass of the native enzyme was estimated to be 88 kDa by size-exclusion chromatography. It seems to be composed of 3 identical subunits with a relative molecular mass of 28 kDa as found by sodium dodecyl sulfate polyacrylamide gel electrophoresis and laser-induced mass spectrometry. The isoelectric point was found to be 4.7-5.0. The optimum temperature is 37 degrees C and the optimum pH for the oxidation and the reduction reaction are 9.0-9.5 and 5.5-6.5, respectively. The purified enzyme was further characterized with respect to substrate specificity, kinetic parameters and amino terminal sequence. Analogues of D(+)-carnitine (L(-)-carnitine, crotonobetaine, gamma-butyrobetaine, carnitine amide, glycine betaine, choline) are competitive inhibitors of D(+)-carnitine oxidation. The equilibrium constant of the reaction of D(+)-carnitine dehydrogenase was determined to be 2.2 x 10(-12). The purified D(+)-carnitine dehydrogenase has similar kinetic properties to the L(-)-carnitine dehydrogenase from the same microorganism as well as to L(-)-carnitine dehydrogenases of other bacteria.
来自土壤杆菌属的D(+)-肉碱脱氢酶催化D(+)-肉碱氧化为3-脱氢肉碱，这是D(+)-肉碱降解的第一步。这种对NAD(+)具有特异性的胞质酶通过4步色谱法纯化了126倍，达到表观电泳均一性。通过尺寸排阻色谱法估计天然酶的分子量为88 kDa。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳和激光诱导质谱分析发现，它似乎由3个相对分子质量为28 kDa的相同亚基组成。发现其等电点为4.7 - 5.0。最适温度为37℃，氧化反应和还原反应的最适pH分别为9.0 - 9.5和5.5 - 6.5。对纯化后的酶在底物特异性、动力学参数和氨基末端序列方面进行了进一步表征。D(+)-肉碱的类似物（L(-)-肉碱、巴豆甜菜碱、γ-丁酸甜菜碱、肉碱酰胺、甘氨酸甜菜碱、胆碱）是D(+)-肉碱氧化的竞争性抑制剂。D(+)-肉碱脱氢酶反应的平衡常数测定为2.2×10(-12)。纯化后的D(+)-肉碱脱氢酶与来自同一微生物的L(-)-肉碱脱氢酶以及其他细菌的L(-)-肉碱脱氢酶具有相似的动力学性质。
