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Analysis of the Nucleotide Binding Sites of Mitochondrial ATP Synthase Provides Evidence for a Two-Site Catalytic Mechanism

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Analysis of the Nucleotide Binding Sites of Mitochondrial ATP Synthase Provides Evidence for a Two-Site Catalytic Mechanism Biochimica et Biophysica Acta 1458 (2000) 234^251 www.elsevier.com/locate/bba Review Analysis of the nucleotide binding sites of mitochondrial ATP synthase provides evidence for a two-site catalytic mechanism J.A. Berden *, A.F. Hartog E.C. Slater Institute, BioCentrum, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands 1. Introduction interactions and the conformational changes during catalysis are studied in much detail, but for conclu- The complexity of the mechanism of ATP hydro- sions on the precise mechanism of catalysis the be- lysis and ATP synthesis by the ATP synthase is illus- haviour and properties of the nucleotide binding trated by the large number of nucleotide binding sites, the topic of this review, have to be taken into sites. They are all localised on the F1 part of the account. enzyme, the hydrophylic moiety that is responsible for the catalytic reaction. Many e¡orts have been 1.1. Well-established features of the ATP synthase made to characterise all the six binding sites and to analyse the role of the nucleotides at each of these Some features of the ATP synthase, considered as sites. In the present paper we will list ¢rst some well well established, may serve as starting point for our established features of the ATP synthase and then analysis. We name the following. discuss the results of studies on the nucleotide bind- (1) F1 contains six nucleotide binding sites, located ing sites of the enzyme and draw some conclusions at each of the three K- and three L-subunits of the on the catalytic mechanism of ATP hydrolysis and enzyme. After the evidence for the presence of three synthesis. We will mainly describe studies with mito- K- and three L-subunits in bacterial F1 [1], it took chondrial F1, but where relevant, studies on the en- some years before the stoichiometry of the subunits zyme from other sources and on the full F0F1 (F0 is of the mitochondrial F1 was established [2,3] and the hydrophobic membrane-embedded part of the again a few years until also suitable evidence for a enzyme) will be mentioned. At present the subunit 3:3 stoichiometry of the large subunits of the chlo- roplast enzyme was provided [4]. Concomitantly the presence of six sites for adenine nucleotides was ¢rmly established for F1 from various sources [5^9]. Abbreviations: 8-N3-AD(T)P, 8-azido-AD(T)P; 2-N3- As far as the nomenclature is concerned, we will call AD(T)P, 2-azido-AD(T)P; AMPPNP, 5P-adenylyl L,Q-imidodi- them K-orL-sites, depending on the mainly contri- phosphate; BzAD(T)P, 3P-O-(4-benzoyl)benzoyl-AD(T)P; buting subunit, although it has been shown in many NAP3-2-N3-ADP, 3P-O-[3-[N-(4-azido-2-nitrophenyl)amino]pro- pionyl]-2-azido-ADP; NbfCl, 7-chloro-4-nitrobenzofurazan; experiments [7,10^13] and con¢rmed by the crystal FSBA, 5P-p-£uorosulfonylbenzoyladenosine; F1, hydrophylic structure [14] that both sites are located at interfaces part of the ATP synthase; F0, hydrophobic membrane-part of between K- and L-subunits. the ATP synthase; F0F1, complete ATP synthase; MF1, mito- (2) The catalytic sites are located on L-subunits. chondrial F1 ;CF1, chloroplast F1 ;TF1,F1 from the thermo- phylic bacterium PS3; SMP, submitochondrial particles This conclusion was already drawn from the initial * Corresponding author. Fax: +31-20-525-5124; studies on nucleotide binding [6,15,16], further estab- E-mail: [email protected] lished by the formulation of a consensus sequence for 0005-2728 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S0005-2728(00)00076-1 BBABIO 44835 22-5-00 J.A. Berden, A.F. Hartog / Biochimica et Biophysica Acta 1458 (2000) 234^251 235 binding site has to be de¢ned with respect to all four relevant aspects: binding a¤nity (tight or loose bind- ing), exchangeability (non-exchangeable, slowly ex- Fig. 1. Schematic representation of the nucleotide binding sites changeable, rapidly exchangeable), catalytic involve- of F1. The upper row represents the three L-sites (1^3) in order of decreasing a¤nity for nucleotides. The lower row represents ment and localisation. the three K-sites (4^6) in order of decreasing a¤nity. The statement that the catalytic sites are coopera- tive may be a useful tool in the analysis of experi- ATPases [17] and ¢nally unequivocally con¢rmed by ments in which several sites are modi¢ed at the same the reported crystallographic data [14]. time with a covalently binding analogue: if the in- (3) The catalytic mechanism of ATP synthase im- hibition curve, i.e., the curve relating activity with plies the cooperative involvement of more than one the number of modi¢ed sites, is linear, maximally catalytic sites and these catalytic sites show negative one catalytic site is involved in the modi¢cation, cooperativity of binding and positive cooperativity of independent of the total number of sites that has to catalysis. The huge mountain of arguments for these be modi¢ed to obtain full inhibition. If more cata- two assumptions have been well described by Boyer lytic sites are involved, the inhibition curve cannot be [18] and they may be considered as established facts. linear. However, this only holds when it is certain (4) Tight binding of a nucleotide at a catalytic site that the covalent modi¢cation itself is not coopera- is an intermediate step in catalysis [19,20]. Upon re- tive. moval of loosely bound nucleotides, one nucleotide It has been well established that the three L-sub- always remains bound at a catalytic site [21]. This units at any time point not only di¡er in binding nucleotide remains tightly bound as long as the other a¤nity, but are also conformationally di¡erent, re- catalytic site(s) are empty. sulting in di¡erent speci¢city of ligands [22] and dif- On the basis of the formulated assumptions, we ferent interaction with the small subunits [23,24]. will try to describe the properties of each of the six This aspect will be treated in other contributions to binding sites and the role of the nucleotides at these this issue and we will restrict ourselves to just the sites in the process of ATP synthesis and hydrolysis. binding properties of each site under speci¢c condi- We will refer to the sites, if appropriate, with a num- tions. ber, as represented in Fig. 1. The three L-sites are numbered 1^3 and the three K-sites 4^6, in order of decreasing a¤nity. 2. Di¡erences between di¡erent F1 preparations and di¡erent forms of F1 1.2. The di¡erent aspects of nucleotide binding 2.1. Di¡erent preparations of mitochondrial F1 When studying the nucleotide binding sites of F1 it is important to di¡erentiate between the various ele- For the correct analysis of experiments in which ments that are relevant for the characterisation of a nucleotides have been bound to the enzyme, have nucleotide binding site. In the introduction to several been exchanged or have been removed, it is impor- papers the error is made that rapid exchangeability is tant to know the starting situation. The most widely identi¢ed with catalytic involvement, tight binding used preparation of bovine heart F1 is isolated ac- with a non-catalytic role and non-exchangeability cording to the procedure of Knowles and Penefsky with localisation on an K-subunit. However, a cata- [25]. Before use the preparation is freed of loosely lytic site may contain a tightly bound nucleotide, a bound nucleotides by ammonium sulfate precipita- non-catalytic site may contain a rapidly exchange- tion and column centrifugation [26] in the presence able nucleotide and a L-subunit may contain a bind- of EDTA. This preparation contains three tightly ing site that is not participating in rapid catalysis, bound nucleotides [5,27^31], but when Mg2 has despite the fact that only L-sites have catalytic po- been added before the removal of free and loosely tential. It is therefore necessary to de¢ne the property bound nucleotides, about four nucleotides remain that is determined in a speci¢c experiment and each bound [29,32]. On the other hand, the preparations BBABIO 44835 22-5-00 236 J.A. Berden, A.F. Hartog / Biochimica et Biophysica Acta 1458 (2000) 234^251 mostly used by the group of Allison [33,34] and the from bacteria, chloroplasts and mammals also re- pig heart enzyme used by the group of Gautheron spond di¡erently towards the activating anion sul¢te, [35,36] contain only two tightly bound nucleotides an inhibitor of ATP synthesis in some preparations, and such enzyme preparations respond di¡erently but not in others [43]. A well-known example is also towards added nucleotides. The most well-known ex- the presence of tightly bound nucleotides. The pres- ample is the so-called hysteretic inhibition after pre- ence of tightly bound nucleotides in bovine heart F1, incubation of the enzyme with ADP plus Mg2 : discovered in the seventies [28,44], was important for upon addition of ATP to ADP-incubated enzyme the development of the idea that catalysis occurs the hydrolysis activity starts at a maximal rate, but when a nucleotide is tightly bound at a catalytic slows down to 15^20% after a few minutes. This site [19,20], but F1 preparations from the thermo- behaviour is not seen when the enzyme is prepared phylic bacterium PS3 do not contain any tightly according to the procedure of Knowles and Penefsky bound nucleotide [1] and the bound nucleotides [25].
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