Functional importance of ?Asp-350 in the catalytic sites of Escherichia coli ATP synthase.

ABSTRACT: Negatively charged residue ?Asp-350 of the highly conserved VISIT-DG sequence is required for Pi binding and maintenance of the phosphate-binding subdomain in the catalytic sites of Escherichia coli F1Fo ATP synthase. ?Asp-350 is situated in close proximity, 2.88?Å and 3.5?Å, to the conserved known phosphate-binding residues ?R376 and ?R182. ?D350 is also in close proximity, 1.3?Å, to another functionally important residue ?G351. Mutation of ?Asp-350 to Ala, Gln, or Arg resulted in substantial loss of oxidative phosphorylation and reduction in ATPase activity by 6- to 16-fold. The loss of the acidic side chain in the form of ?D350A, ?D350Q, and ?D350R caused loss of Pi binding. While removal of Arg in the form of ?R376D resulted in the loss of Pi binding, the addition of Arg in the form of ?G351R did not affect Pi binding. Our data demonstrates that ?D350R helps in the proper orientation of ?R376 and ?R182 for Pi binding. Fluoroaluminate, fluoroscandium, and sodium azide caused almost complete inhibition of wild type enzyme and caused variable inhibition of ?D350 mutant enzymes. NBD-Cl (4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole) caused complete inhibition of wild type enzyme while some residual activity was left in mutant enzymes. Inhibition characteristics supported the conclusion that NBD-Cl reacts in ?E (empty) catalytic sites. Phosphate protected against NBD-Cl inhibition of wild type and ?G351R mutant enzymes but not inhibition of ?D350A, ?D350Q, ?D350R, or ?R376D mutant enzymes. These results demonstrate that ?Asp-350 is an essential residue required for phosphate binding, through its interaction with ?R376 and ?R182, for normal function of phosphate binding subdomain and for transition state stabilization in ATP synthase catalytic sites.

SUBMITTER: Raheem S 

PROVIDER: S-EPMC6718308 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature