Catalytic properties of Escherichia coli F1-ATPase depleted of endogenous nucleotides.
Nucleotide-depleted Escherichia coli F1 was prepared by the procedure of Wise et al. (1983, Biochem. J. 215, 343-350). This enzyme had high rates of steady-state ATPase and GTPase activity. When "unisite" ATP hydrolysis was measured using an F1/ATP concentration ratio of 10, all of the substoichiometric ATP became bound to the high-affinity catalytic site and none became bound to noncatalytic sites. The association rate constant for ATP binding was 7 x 10(5) M-1 s-1 and the KdATP was 7.9 x 10(-10) M, as compared to values of 3.8 x 10(5) M-1 s-1 and 1.9 x 10(-10) M, respectively, in native (i.e., nucleotide-replete) F1. Rate constants for bound ATP hydrolysis, ATP resynthesis, and P(i) release, and the reaction equilibrium constant, were similar in nucleotide-depleted and native F1. Therefore, we conclude that occupancy of the noncatalytic sites is not required for formation of the high-affinity catalytic site of F1 and has no significant effect on unisite catalysis. In further experiments we looked for the occurrence of inhibitory, catalytic-site-bound MgADP in E. coli F1. Such an entity has been reported for chloroplast and mitochondrial F1. However, our experiments gave no indication for inhibitory MgADP in E. coli F1.
Senior
A E
AE
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
Lee
R S
RS
al-Shawi
M K
MK
Weber
J
J
D000244:Adenosine Diphosphate / Q000032:analysis / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000032:analysis / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D020558:GTP Phosphohydrolases; D006180:Proton-Translocating ATPases