Ching M H, Kaur P, Karkaria C E, Steiner R F, Rosen B P
Department of Biochemistry, Wayne State University, School of Medicine, Detroit, Michigan 48201.
J Biol Chem. 1991 Feb 5;266(4):2327-32.
The ArsA protein, the catalytic component of the plasmid-encoded resistance system for removal of the toxic oxyanions arsenite, antimonite, and arsenate from bacterial cells, catalyzes oxyanion-stimulated ATP hydrolysis. Three lines of evidence suggest that the ArsA protein functions as a homodimer. First, the ArsA protein was modified with 5'-p-fluorosulfonyl-benzoyladenosine (FSBA). Antimonite potentiated FSBA inhibition, while ATP or ADP afforded partial protection. ATP and antimonite together provided complete protection, indicating interaction of the anion- and nucleotide-binding sites. The estimated Ki values for FSBA were 0.4 mM in the absence of antimonite and 0.1 mM in the presence of antimonite, suggesting that the binding of antimonite increased the affinity of ArsA protein for FSBA. Incorporation of [14C]FSBA was examined. Extrapolation of the amount of FSBA required to inactivate the protein indicated that 1 mol of FSBA was sufficient to inhibit the activity of 1 mol of ArsA protein in the absence of substrates, while only 0.5 mol was required in the presence of the anionic substrate antimonite. Second, chemical cross-linking of the 63-kDa ArsA protein with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline resulted in formation of a species approximately twice the size of the monomer in the presence of antimonite but not ATP. Third, determination of the average mass of the ArsA protein in solution by light scattering demonstrated that the average species was 66 kDa in the absence of substrates. In the presence of antimonite the weight average molecular mass increased to a mass in excess of 100 kDa. These results are consistent with the ArsA protein existing in an equilibrium between monomer and dimer, with the equilibrium favoring dimerization upon binding of the anionic substrate. Moreover, total loss of ATPase activity in the half-modified enzyme suggests that the catalytic sites on each monomer must interact.
ArsA蛋白是质粒编码的抗性系统的催化成分，该系统用于从细菌细胞中去除有毒的氧阴离子亚砷酸盐、亚锑酸盐和砷酸盐，它催化氧阴离子刺激的ATP水解。有三条证据表明ArsA蛋白以同型二聚体的形式发挥作用。首先，用5'-对氟磺酰基苯甲酰腺苷（FSBA）修饰ArsA蛋白。亚锑酸盐增强了FSBA的抑制作用，而ATP或ADP提供了部分保护。ATP和亚锑酸盐共同提供了完全保护，表明阴离子结合位点和核苷酸结合位点之间存在相互作用。在不存在亚锑酸盐的情况下，FSBA的估计Ki值为0.4 mM，在存在亚锑酸盐的情况下为0.1 mM，这表明亚锑酸盐的结合增加了ArsA蛋白对FSBA的亲和力。检测了[14C]FSBA的掺入情况。外推使蛋白失活所需的FSBA量表明，在没有底物的情况下，1摩尔FSBA足以抑制1摩尔ArsA蛋白的活性，而在存在阴离子底物亚锑酸盐的情况下，仅需0.5摩尔。其次，63 kDa的ArsA蛋白与N-乙氧基羰基-2-乙氧基-1,2-二氢喹啉进行化学交联，在存在亚锑酸盐而不是ATP的情况下，形成了一种大小约为单体两倍的物质。第三，通过光散射测定溶液中ArsA蛋白的平均质量，结果表明在没有底物的情况下，平均物质为66 kDa。在存在亚锑酸盐的情况下，重均分子量增加到超过100 kDa。这些结果与ArsA蛋白以单体和二聚体之间的平衡形式存在一致，该平衡在阴离子底物结合时有利于二聚化。此外，半修饰酶中ATPase活性的完全丧失表明每个单体上的催化位点必须相互作用。
