| Abstrakt: |
The ATP synthase of E. coli requires for function a conserved residue R210 in the membrane-bound, stator subunit a. Recent work has shown that ATP synthesis can be maintained if the conserved Arg is switched with a conserved Gin at position 252 in an adjacent transmembrane helix. Furthermore, in this construct (R210Q/Q252R), but not in the wild type, Lys can replace the Arg with retention of function. In both cases, the activity is increased if a third mutation is introduced: P204T. Mutagenesis of P204 to either Ala or Ser was also effective, indicating that replacement of the Pro is sufficient for enhancement of the activity of R210Q/Q252R. Saturation mutagenesis was applied to the 210 position in the background of P204T/Q252R. The results showed that only a limited number of substitutions permitted function, indicating that both positions, 210 and 252, are important. Finally, E219 was mutagenized in the background of the triple mutant (P204T/R210Q/Q252R). These results indicated that while the triple mutant is capable of ATP synthesis, the proton pathway through subunit a is likely altered. [ABSTRACT FROM AUTHOR] |
Plný text