Studies of nucleotide binding to the catalytic sites of Escherichia coli βY331W-F ₁ -ATPase using fluorescence quenching

Abstract

ATP synthase ͉ bi-site catalysis ͉ phosphate ͉ sulfate ͉ tri-site catalysis D uring oxidative and photophosphorylation, F o F 1 -ATP synthase is responsible for reversible synthesis of ATP coupled to transmembrane movement of H ϩ (or, in some bacteria, Na ϩ ). The enzyme can be separated into two multisubunit components, the membranous factor F o , which in bacteria consists of a ring of c subunits and an a and b subunit in a stoichiometry of ab 2 c n , and the soluble factor F 1 with a subunit composition of ␣ 3 ␤ 3 ␥␦. Energy transformation by F o F 1 is best described by the binding change mechanism (1) and involves a rotation of a complex of subunits (␥c n , rotor) within the rest of the enzyme (stator) (2, 3). F 1 is the catalytic component of ATP synthase and, when isolated, is capable only of net ATP hydrolysis. In the crystal structure of F 1 from beef heart mitochondria (MF 1 ), six nucleotide-binding sites are located at the interfaces between ␣ and ␤ subunits that are arranged alternately around an asymmetrical ␣-helical-coiled coil formed by ␥-subunit (4). Three of the nucleotide-binding sites formed mostly by side chains of ␤ subunits are catalytic, and another three primarily on ␣ subunits are noncatalytic (5).Three different conformations of ␤ subunits in the crystal structure of MF 1 are associated with asymmetric interactions with the ␥ subunit and are thought to represent distinct states that each of the catalytic sites sequentially assumes during catalytic cycle according to the binding change mechanism (4). That the ␥ subunit plays a crucial role in determining properties of the catalytic sites is supported by the crystal structure of ␣ 3 ␤ 3 -subcomplex of F 1 from the thermophilic Bacillus PS3 (TF 1 ). In this structure, all of the ␤ subunits are in very similar conformations, and the three catalytic sites are in a state similar to one of the three states (open) found in the crystal structure of MF 1 (6). In addition, the strong positive catalytic cooperativity in catalysis by F 1 (7,8) that is mediated by a rotation of ␥ subunit (9, 10) is lacking in the ␣ 3 ␤ 3 -subcomplex. Interactions between ␣ and ␤ subunits are also considered as contributing to the modulation of the state of catalytic sites in F 1 (11,12).Whether asymmetric interactions of the three ␤ subunits with ␥, together with ␣/␤ interactions, are sufficient to induce asymmetry in the properties of the catalytic sites is not clear. According to Senior and colleagues (13-15), intersubunit interactions in F 1 alone cannot induce nucleotide-binding heterogeneity at catalytic sites. Based on observations obtained with ␤Y331W-mutant F 1 from Escherichia coli (EcF 1 ), it has been argued that in the absence of Mg 2ϩ , all three catalytic sites bind nucleotides with identical and low affinity and are in a similar state. Mg 2ϩ is considered to play a crucial role in inducing asymmetry between catalytic sites, with high-affinity nucleotide binding at a single catalytic site. This role, according to (15), extends beyond Mg 2ϩ being simpl...

Conformational rearrangements of the C1 ring in KaiC measure the timing of assembly with KaiB

“…In fact (Fig. 4b ), one edge of the indole ring of W229 is in close proximity to potential quenchers, including the adenine ring of bound ATP 19 and a main-chain oxygen atom of I239 20 , and the other edge is nearly in contact with H33 21 . Collectively, these results indicate that the C1 ring undergoes a structural change while maintaining its hexameric structure.…”

Temperature Dependence of the Rotation and Hydrolysis Activities of F1-ATPase

“…The scheme above for the timing of ADP release at 23°C is based on the behavior of a fluorescent ATP analog (11), and may not apply to reactions involving unlabeled ATP. There have been reports that suggest that ADP release occurs before ATP binding and thus F 1 operates in the socalled bisite scheme where the nucleotide occupancy of the three catalytic sites alternates between one and two (27,28) as opposed to two and three (mostly two) in our scheme (11) or in an earlier proposal (29). This site-occupancy issue is yet unsettled, and possibilities other than the rate-limiting process being ADP release cannot be ruled out.…”

Energetic Effects of Magnesium in the Recognition of Adenosine Nucleotides by the F1-ATPase β Subunit

“…In spite of the wealth of available information about the catalytic and binding properties of F 1 , there is still a debate regarding whether the occupancy of two ,,, or three catalytic sites ,, is sufficient to achieve rapid ATPase activity. Furthermore, binding and kinetic data of ATP synthase and its different subcomplexes have proven to be difficult to analyze and interpret.…”

“…Besides its role as a cofactor required for stabilization of the transition state , Mg(II) has been implicated as being crucial in determining the binding asymmetry and cooperativity between catalytic sites , . However, recent studies have challenged that conclusion, indicating that heterogeneity in β subunit affinities is an inherent property of nucleotide-free F 1 ,, . As far as we know, no systematic characterization of the energetic contribution of Mg(II) in the recognition of nucleotides by F 1 or by the isolated subunits has been conducted.…”