Li J, Rosen B P
Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
J Biol Chem. 1998 Mar 20;273(12):6796-800. doi: 10.1074/jbc.273.12.6796.
ArsA, the catalytic subunit of an anion-translocating ATPase, has two consensus nucleotide binding sites, one N-terminal and one C-terminal. A mutation producing a G15C substitution in the N-terminal domain resulted in substantial reductions in arsenite resistance, transport, and ATPase activity. A second site revertant (A344V) adjacent to the C-terminal nucleotide binding site was previously shown to restore arsenite resistance, suggesting the interaction of the nucleotide binding sites in ArsA (Li, J., Liu, S., and Rosen, B. P. (1996) J. Biol. Chem. 271, 25247-25252). In this study, it is shown that alteration of Ala-344 to bulkier residues, including Cys, Thr, Pro, Asp, Leu, Phe, Tyr, or Arg, also suppressed the G15C substitution. However, A344G or A344S substitutions only marginally suppressed the primary mutation. Alteration of Gly-15 to Ala, Cys, Asp, Tyr, or Arg each resulted in decreased arsenite resistance. The larger the residue volume of the substitution, the lower the resistance, with a G15R substitution producing the least resistance. Resistance in a strain expressing an arsA gene encoding the G15R substitution could be rescued by A344S, A344T, A344D, A344R, or A344V second site suppressors. The larger the residue is then the greater the suppression is. The in vitro ArsA ATPase activities from purified wild type, G15A, G15C, and G15R exhibits an inverse relationship between activity and residue volume. Purified G15A and G15C exhibited both an increase in the Km for ATP and a decrease in Vmax. The results are consistent with a physical interaction of the two nucleotide binding domains and indicate that the geometry at the interface between the N- and C-terminal nucleotide binding sites places spatial constraints on allowable residues in that interface.
阴离子转运ATP酶的催化亚基ArsA有两个共有核苷酸结合位点，一个在N端，一个在C端。在N端结构域产生G15C替换的突变导致亚砷酸盐抗性、转运和ATP酶活性大幅降低。先前显示，与C端核苷酸结合位点相邻的第二个位点回复突变体（A344V）可恢复亚砷酸盐抗性，这表明ArsA中核苷酸结合位点之间存在相互作用（Li, J., Liu, S., and Rosen, B. P. (1996) J. Biol. Chem. 271, 25247 - 25252）。在本研究中，结果表明，将Ala - 344替换为更大体积的残基，包括Cys、Thr、Pro、Asp、Leu、Phe、Tyr或Arg，也能抑制G15C替换。然而，A344G或A344S替换仅略微抑制了初级突变。将Gly - 15替换为Ala、Cys、Asp、Tyr或Arg均导致亚砷酸盐抗性降低。替换残基的体积越大，抗性越低，其中G15R替换产生的抗性最低。表达编码G15R替换的arsA基因的菌株中的抗性可被A344S、A344T、A344D、A344R或A344V第二个位点抑制子挽救。残基越大，抑制作用越强。纯化的野生型、G15A、G15C和G15R的体外ArsA ATP酶活性与残基体积之间呈反比关系。纯化的G15A和G15C对ATP的Km均增加，Vmax均降低。这些结果与两个核苷酸结合结构域的物理相互作用一致，并表明N端和C端核苷酸结合位点之间界面处的几何形状对该界面中允许的残基施加了空间限制。
