A more robust version of the Arginine 210-switched mutant in subunit a of the Escherichia coli ATP synthase.
Previous work has shown that the essential R210 of subunit a in the Escherichia coli ATP synthase can be switched with a conserved glutamine Q252 with retention of a moderate level of function, that a third mutation P204T enhances this function, and that the arginine Q252R can be replaced by lysine without total loss of activity. In this study, the roles of P204T and R210Q were examined. It was concluded that the threonine in P204T is not directly involved in function since its replacement by alanine did not significantly affect growth properties. Similarly, it was concluded that the glutamine in R210Q is not directly involved with function since replacement by glycine results in significantly enhanced function. Not only did the rate of ATP-driven proton translocation increase, but also the sensitivity of ATP hydrolysis to inhibition by N,N'-dicyclohexylcarbodiimide (DCCD) rose to more than 50%. Finally, mutations at position E219, a residue near the proton pathway, were used to test whether the Arginine-switched mutant uses the normal proton pathway. In a wild type background, the E219K mutant was confirmed to have greater function than the E219Q mutant, as has been shown previously. This same unusual result was observed in the triple mutant background, P204T/R210Q/Q252R, suggesting that the Arginine-switched mutants are using the normal proton pathway from the periplasm.
Southern Methodist University, Department of Biological Sciences, Dallas, TX 75275-0376, USA.
Leon
L
Bae
Steven B
SB
Vik
D001120:Arginine / Q000235:genetics / Q000502:physiology*; D025243:Bacterial Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005973:Glutamine / Q000235:genetics / Q000502:physiology; D006868:Hydrolysis / Q000187:drug effects; D016296:Mutagenesis; D009154:Mutation; D013329:Structure-Activity Relationship
D005973:Glutamine; D004024:Dicyclohexylcarbodiimide; D001120:Arginine; D025243:Bacterial Proton-Translocating ATPases