Epsilon subunit of Escherichia coli F1-ATPase: effects on affinity for aurovertin and inhibition of product release in unisite ATP hydrolysis.
The epsilon subunit of Escherichia coli F1-ATPase is a tightly bound but dissociable partial inhibitor of ATPase activity. The effects of epsilon on the enzyme were investigated by comparing the ATPase activity and aurovertin binding properties of the epsilon-depleted F1-ATPase and the epsilon-replete complex. Kinetic data of multisite ATP hydrolysis were analyzed to give the best fit for one, two, or three kinetic components. Each form of F1-ATPase contained a high-affinity component, with a Km near 20 microM and a velocity of approximately 1 unit/mg. Each also exhibited a component with a Km in the range of 0.2 mM. The velocity of this component was 25 units/mg for epsilon-depleted ATPase but only 4 units/mg for epsilon-replete enzyme. The epsilon-depleted enzyme also contained a very low affinity component not present in the epsilon-replete enzyme. In unisite hydrolysis studies, epsilon had no effect on the equilibrium between substrate ATP and product ADP.P1 at the active site but reduced the rate of product release 15-fold. These results suggest that epsilon subunit slows a conformational change that is required to reduce the affinity at the active site, allowing dissociation of product. It is suggested that inhibition of multisite hydrolysis by epsilon is also due to a reduced rate of product release. epsilon-depleted F1-ATPase showed little of no modulation of aurovertin fluorescence by added ADP and ATP. Aurovertin fluorescence titrations in buffer containing ethylenediaminetetraacetic acid (EDTA) revealed that epsilon-depleted enzyme had high affinity for aurovertin (Kd less than 0.1 microM) regardless of the presence of nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)
D000255:Adenosine Triphosphate / Q000378:metabolism; D001313:Aurovertins / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011714:Pyrans / Q000378:metabolism*
D001313:Aurovertins; D046911:Macromolecular Substances; D011714:Pyrans; C039026:aurovertin D; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases