Bhattacharjee H, Li J, Ksenzenko M Y, Rosen B P
Department of Biochemistry, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
J Biol Chem. 1995 May 12;270(19):11245-50. doi: 10.1074/jbc.270.19.11245.
The ArsA protein, the catalytic subunit of the oxyanion-translocating ATPase responsible for resistance to arsenicals and antimonials in Escherichia coli, is activated by arsenite or antimonite. Activation is associated with dimerization of the ArsA protein. Enzymatic activity was rapidly but reversibly inhibited by the sulfhydryl reagent methyl methanethiosulfonate, suggesting that at least one cysteinyl residue is required for catalytic activity. Each of the four cysteinyl residues in the ArsA protein, Cys26, Cys113, Cys172, and Cys422, were individually changed to seryl residues. The C26S protein had normal properties. Cells expressing the other three mutations lost resistance to arsenite and antimonite. The C113S, C172S, and C422S enzymes each had relatively normal Km values for ATP but reduced affinity for antimonite and arsenite. The Vmax of the activated enzymes ranged from very low for the C113S and C422S enzymes to near normal for the C172S enzyme. These results suggest a mechanism of activation by formation of a tricoordinate complex between Sb(III) or As(III) and the cysteine thiolates 113, 172, and 422.
ArsA蛋白是大肠杆菌中负责抗砷和抗锑的氧阴离子转运ATP酶的催化亚基，可被亚砷酸盐或亚锑酸盐激活。激活与ArsA蛋白的二聚化有关。巯基试剂甲硫基甲烷磺酸盐可快速但可逆地抑制酶活性，这表明催化活性至少需要一个半胱氨酸残基。ArsA蛋白中的四个半胱氨酸残基Cys26、Cys113、Cys172和Cys422分别被突变为丝氨酸残基。C26S蛋白具有正常特性。表达其他三种突变的细胞失去了对亚砷酸盐和亚锑酸盐的抗性。C113S、C172S和C422S酶对ATP的Km值相对正常，但对亚锑酸盐和亚砷酸盐的亲和力降低。激活酶的Vmax范围从C113S和C422S酶的非常低到C172S酶的接近正常。这些结果表明了一种激活机制，即Sb(III)或As(III)与半胱氨酸硫醇盐113、172和422之间形成三配位复合物。
