Suh S, Escalante-Semerena J C
Department of Bacteriology, University of Wisconsin-Madison 53706-1567.
J Bacteriol. 1995 Feb;177(4):921-5. doi: 10.1128/jb.177.4.921-925.1995.
The cobA gene of Salmonella typhimurium and its product were overexpressed to approximately 20% of the total cell protein. CobA was purified to 98% homogeneity; N-terminal sequence analysis (21 residues) of homogeneous protein confirmed the predicted amino acid sequence. ATP:corrinoid adenosyltransferase activity was demonstrated in vitro to be associated with CobA. This activity was optimal at pH 8 and 37 degrees C. A quantitative preference was determined for Mn(II) cations and ATP. The apparent Km of CobA for ATP was 2.8 microM, and that for cob(I)alamin was 5.2 microM. Vmax was measured at 0.43 nmol/min. Cobinamide served as the substrate for CobA to yield adenosylcobinamide. Activity was stable at 4 degrees C for several weeks but was lost rapidly at room temperature (50% overnight). Dithiothreitol was required to maintain the enzymatic activity of CobA.
鼠伤寒沙门氏菌的cobA基因及其产物的表达量在总细胞蛋白中约占20%。CobA被纯化至98%的纯度；对纯化后的蛋白质进行N端序列分析（21个残基），证实了预测的氨基酸序列。体外实验证明，ATP：类咕啉腺苷基转移酶活性与CobA相关。该活性在pH 8和37℃时最佳。确定了对Mn(II)阳离子和ATP存在定量偏好。CobA对ATP的表观Km为2.8 μM，对钴胺素（I）的表观Km为5.2 μM。Vmax测得为0.43 nmol/min。钴胺酰胺作为CobA的底物生成腺苷钴胺酰胺。该活性在4℃下可稳定数周，但在室温下会迅速丧失（一夜之间丧失50%）。需要二硫苏糖醇来维持CobA的酶活性。
