Reactions of a Fluorescent ATP Analog, 2′-(5-Dimethyl-Aminonaphtha lene-1-Sulfonyl) Amino-2′-DeoxyATP, with E. coli F1-ATPase and Its Subunits: The Roles of the High Affinity Binding Site in the α Subunit and the Low Affinity Binding Site in the β Subunit1

Abstract

We performed kinetic studies on the reactions of a fluorescent ATP analog, 2′-(5-dimethyl-aminonaphthalene-1-sulfonyl) amino-2′-deoxyATP (DNS-ATP), with E. coli F₁-ATPase (EF₁) and its subunits, to clarify the role of each subunit in the ATPase reaction. The following results were obtained. 1. One mol of EF₁, which contains nonexchangeable 2 mol ATP and 0.5 mol ADP, binds 3 mol of DNS-ATP. The apparent dissociation constant, in the presence of Mg²⁺, was 0.23 μM. Upon binding, the fluorescence intensity of DNS-ATP at 520 nm increased exponentially with t_1/2 of 35 s, and reached 3.5 times the original fluorescence level. Following the fluorescence increase, DNS-ATP was hydrolyzed, and the fluorescence intensity maintained its enhanced level. 2. The addition of an excess of ATP over the EF₁-DNS-nucleotide complex, in the presence of Mg²⁺, decreased the fluorescence intensity rapidly, indicating the acceleration of DNS-nucleotide release from EF₁. ADP and GTP also decreased the fluorescence intensity. 3. DCCD markedly inhibited the accelerating effect of ATP on DNS-nucleotide release from EF₁ and the EF₁-DNS-ATPase or -ATPase activity in a steady state. On the other hand, DCCD only slightly inhibited the fluorescence increase of DNS-ATP, due to its binding to EF₁, and the rate of single cleavage of 1 mol of DNS-ATP per mol of a subunit of EF₁. 4. In the presence of Mg²⁺, 0.65–0.82 mol of DNS-ATP binds to 1 mol of the isolated a subunit of EF1 with an apparent dissociation constant of 0.06–0.07 μM. Upon binding, the fluorescence intensity of DNS-ATP at 520 nm increased 1.55 fold very rapidly (t_1/2 < 1 s). No hydrolysis of DNS-ATP was observed upon the addition of the isolated a subunit. The fluorescence intensity of DNS-ATP was unaffected by the addition of the isolated β subunit. DNS-ATP was also unhy-drolyzed by the isolated β subunit. 5. EF₁-ATPase was reconstituted from α, β, and γ subunits in the presence of Mg²⁺ and ATP. The kinetic properties of the fluorescence change of DNS-ATP in the reaction with the reconstituted EF₁-ATPase were quite similar to those of native EF₁. Most of our findings are consistent with a simple mechanism that the high affinity catalytic site and low affinity regulatory site exist in the α subunit and β subunit, respectively. However, the findings mentioned in (4) suggest that the binding of the α and β subunit, which is mediated by the γ subunit, induces con-formational change(s) in the ATP binding site located probably in the a subunit, and that the conformational changes) is essential to exert the full hydrolyzing activity.