Effects of mutations of conserved Lys-155 and Thr-156 residues in the phosphate-binding glycine-rich sequence of the F1-ATPase beta subunit of Escherichia coli.

beta Lys-155 in the glycine-rich sequence of the beta subunit of Escherichia coli F1-ATPase has been shown to be near the gamma-phosphate moiety of ATP by affinity labeling (Ida, K., Noumi, T., Maeda, M., Fukui, T., and Futai, M. (1991) J. Biol. Chem. 266, 5424-5429). For examination of the roles of beta Lys-155 and beta Thr-156, mutants (beta Lys-155-->Ala, Ser, or Thr; beta Thr-156-->Ala, Cys, Asp, or Ser; beta Lys-155/beta Thr-156-->beta Thr-155/beta Lys-156; and beta Thr-156/beta Val-157-->beta Ala-156/beta Thr-157) were constructed, and their properties were studied extensively. The beta Ser-156 mutant was active in ATP synthesis and had approximately 1.5-fold higher membrane ATPase activity than the wild type. Other mutants were defective in ATP synthesis, had < 0.1% of the membrane ATPase activity of the wild type, and showed no ATP-dependent formation of an electrochemical proton gradient. The mutants had essentially the same amounts of F1 in their membranes as the wild type. Purified mutant enzymes (beta Ala-155, beta Ser-155, beta Ala-156, and beta Cys-156) showed low rates of multisite (< 0.02% of the wild type) and unisite (< 1.5% of the wild type) catalyses. The k1 values of the mutant enzymes for unisite catalysis were lower than that of the wild type: not detectable with the beta Ala-156 and beta Cys-156 enzymes and 10(2)-fold lower with the beta Ala-155 and beta Ser-155 enzymes. The beta Thr-156-->Ala or Cys enzyme showed an altered response to Mg2+, suggesting that beta Thr-156 may be closely related to Mg2+ binding. These results suggest that beta Lys-155 and beta Thr-156 are essential for catalysis and are possibly located in the catalytic site, although beta Thr-156 could be replaced by a serine residue.     

Site-directed mutagenesis of stable adenosine triphosphate synthase

Evidence was obtained that four ionizable residues in the alpha and beta subunits of thermophilic ATP synthase (TF0F1), corresponding to Lys-21 and Asp-119 in the MgATP binding segments of adenylate kinase, are essential for the normal catalytic activity. TF0F1 was used because it is the only ATP synthase whose alpha-, beta- and gamma-subunits can be reassembled into an active complex in the absence of both ATP and Mg. Lys-164 and Asp-252 of its beta-subunit were modified to isoleucine and asparagine, respectively, by site-directed mutagenesis using a multifunctional plasmid, and these genes were over-expressed in Escherichia coli. The resulting beta I164 and beta N252 subunits were both noncatalytic after re-assembly into the alpha beta gamma-complex, even though both subunits bound significant amounts of ADP. When Lys-175 and Asp-261 of the alpha-subunit were similarly replaced by isoleucine and asparagine, respectively, the resulting alpha I175 subunit reassembled weakly into an oligomer, while the alpha N261 subunit showed an increased dissociation constant for ADP and was reconstituted into an alpha beta gamma-complex that showed no inter-subunit cooperativity.     

Loss of unisite and multisite catalyses by Escherichia coli F1 through modification with adenosine tri- or tetraphosphopyridoxal

Pyridoxal phosphate (PLP) and adenosine diphospho (AP2-PL)-, triphospho (AP3-PL)-, and tetraphospho (AP4-PL)-pyridoxals (Tagaya, M., and Fukui, T. (1986) Biochemistry 25, 2958-2964) were tested as potential affinity probes for F1 ATPase of Escherichia coli. Both AP3-PL and AP4-PL bound and inhibited F1 ATPase, whereas PLP and AP2-PL were weak inhibitors. The concentrations of AP3-PL and AP4-PL for half-maximal inactivations of the multisite (steady state) ATPase activity were both 18 microM. The binding of these reagents to a reactive lysyl residue(s) was confirmed from the difference absorption spectra, and the stoichiometry of binding of [3H]AP3-PL to F1 at the saturating level was about 1 mol/mol F1. The analogue bound to both the alpha subunit (about two-thirds of the radioactivity) and the beta subunit (about one-third of the radioactivity). No inactivation of multisite ATPase activity or binding of AP3-PL was observed in the presence of ATP. F1 modified with about one mol of AP3-PL had essentially no uni- and multisite hydrolysis of ATP. The rate of binding of ATP decreased to 10(-2) of that of unmodified F1, and the rate of release of ATP was about two times faster. The equilibrium F1 X ATP in equilibrium F1 X ADP X Pi was shifted toward F1 X ATP, and no promotion of ATP hydrolysis at unisite was observed with excess ATP. These results suggest that the AP3-PL or AP4-PL bound to an active site, and catalysis by the two remaining sites was completely abolished.     

The structure of bovine F1-ATPase inhibited by ADP and beryllium fluoride

The structure of bovine F1-ATPase inhibited with ADP and beryllium fluoride at 2.0 angstroms resolution contains two ADP.BeF3- complexes mimicking ATP, bound in the catalytic sites of the beta(TP) and beta(DP) subunits. Except for a 1 angstrom shift in the guanidinium of alphaArg373, the conformations of catalytic side chains are very similar in both sites. However, the ordered water molecule that carries out nucleophilic attack on the gamma-phosphate of ATP during hydrolysis is 2.6 angstroms from the beryllium in the beta(DP) subunit and 3.8 angstroms away in the beta(TP) subunit, strongly indicating that the beta(DP) subunit is the catalytically active conformation. In the structure of F1-ATPase with five bound ADP molecules (three in alpha-subunits, one each in the beta(TP) and beta(DP) subunits), which has also been determined, the conformation of alphaArg373 suggests that it senses the presence (or absence) of the gamma-phosphate of ATP. Two catalytic schemes are discussed concerning the various structures of bovine F1-ATPase.     

Nucleotide-dependent and dicyclohexylcarbodiimide-sensitive conformational changes in the epsilon subunit of Escherichia coli ATP synthase.

The rate of trypsin cleavage of the epsilon subunit of Escherichia coli F1F0 (ECF1F0) is shown to be ligand-dependent as measured by Western analysis using monoclonal antibodies. The cleavage of the epsilon subunit was rapid in the presence of ADP alone, ATP + EDTA, or AMP-PNP + Mg2+, but slow when Pi was added along with ADP + Mg2+ or when ATP + Mg2+ was added to generate ADP + Pi (+Mg2+) in the catalytic site. Trypsin treatment of ECF1Fo was also shown to increase enzymic activity on a time scale corresponding to that of the cleavage of the epsilon subunit, indicating that the epsilon subunit inhibits ATPase activity in ECF1Fo. The ligand-dependent conformational changes in the epsilon subunit were also examined in cross-linking experiments using the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC). In the presence of ATP + Mg2+ or ADP + Pi + Mg2+, the epsilon subunit cross-linked product was much reduced. Prior reaction of ECF1Fo with dicyclohexylcarbodiimide (DCCD), under conditions in which only the Fo part was modified, blocked the conformational changes induced by ligand binding. When the enzyme complex was reacted with DCCD in ATP + EDTA, the cleavage of the epsilon subunit was rapid and yield of cross-linking of beta to epsilon subunit low, whether trypsin cleavage was conducted in ATP + EDTA or ATP + Mg2+. When enzyme was reacted with DCCD in ATP + Mg2+, cleavage of the epsilon subunit was slow and yield of cross-linking of beta to epsilon high, under all nucleotide conditions for proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity labeling of aminoacyl-tRNA synthetases with adenosine triphosphopyridoxal: probing the Lys-Met-Ser-Lys-Ser signature sequence as the ATP-binding site in Escherichia coli methionyl-and valyl-tRNA synthetases

Pyridoxal 5'-triphospho-5'-adenosine (AP3-PL), the affinity labeling reagent specific for lysine residues in the nucleotide-binding site of several enzymes [Tagaya, M., & Fukui, T. (1986) Biochemistry 25, 2958-2964; Yagami, T., Tagaya, M., & Fukui, T. (1988) FEBS Lett. 229, 261-264], was used to identify the ATP-binding site of Escherichia coli methionyl-tRNA synthetase (MetRS). Incubation of this enzyme with AP3-PL followed by reduction with sodium borohydride resulted in a rapid inactivation of both the tRNA(Met) aminoacylation and the methionine-dependent ATP-PPi exchange activities. Complete inactivation corresponded to the incorporation of 0.98 mol of AP3-PL/mol of monomeric trypsin-modified MetRS. ATP or MgATP protected the enzyme from inactivation. The labeling with AP3-PL was also applied to E. coli valyl-tRNA synthetase (ValRS). Both the tRNA(Val) aminoacylation and the valine-dependent ATP-PPi exchange activities were abolished by the incorporation of 0.91 mol of AP3-PL/mol of monomeric ValRS. AP3-PL was found attached to lysine residues 335, 402, and 528 in the primary structure of MetRS. In the case of ValRS, the AP3-PL-labeled residues corresponded to lysines 557, 593, and 909. We therefore conclude that these lysines of MetRS and ValRS are directed toward the ATP-binding site of these synthetases, more specifically at or close to the subsite for the gamma-phosphate of ATP. AP3-PL-labeled Lys-335 of MetRS and Lys-557 of ValRS belong to the consensus tRNA CCA-binding Lys-Met-Ser-Lys-Ser sequence [Hountondji, C., Dessen, P., & Blanquet, S. (1986) Biochimie 68, 1071-1078].(ABSTRACT TRUNCATED AT 250 WORDS)     

The ATP-binding site in gamma subunit of phosphorylase kinase

To reveal the structure of the ATP-binding site(s) in rabbit muscle phosphorylase kinase, we modified the enzyme with adenosine polyphosphopyridoxals. Adenosine tri- and tetraphosphopyridoxals at micromolar concentrations effectively inactivated the enzyme in a time-dependent manner. Inactivation of the enzyme was accelerated by the addition of Ca2+ and Mg2+. Protection from inactivation was afforded by adenylyl beta,gamma-imidodiphosphate and ADP. In reversible inhibition kinetics, adenosine polyphosphopyridoxals as well as their reduced compounds (adenosine polyphosphopyridoxines) competed with ATP. These results suggest that adenosine polyphosphopyridoxals bind to the ATP-binding site(s) in phosphorylase kinase. When phosphorylase kinase was incubated with adenosine triphosphopyridoxal in the presence of Ca2+ and Mg2+, incorporation of the label into alpha, beta, and gamma subunits was observed. In the absence of both cations, larger amounts of the label were incorporated into all the subunits. Structural study on adenosine triphosphopyridoxal-modified sites in the gamma subunit (having a catalytic site) revealed that Lys-151 is mainly labeled. Based on the results of the present and other studies, it is suggested that the site around Lys-151 is involved in recognition of the substrate protein.     

Binding of Mg2+ to the beta subunit or F1 of H+-ATPase from Escherichia coli

The bindings of Mg2+ to the F1 portion of Escherichia coli H+-ATPase and its isolated alpha and beta subunits were studied with 8-anilinonaphthalene-1-sulfonate (ANS). The fluorescence of ANS increased upon addition of F1 or its alpha subunit or beta subunit, as reported previously (M. Hirano, K. Takeda, H. Kanazawa, and M. Futai (1984) Biochemistry 23, 1652-1656). The fluorescence of ANS bound to F1 or its beta subunit increased significantly with further addition of Mg2+, whereas that of the alpha subunit increased only slightly. Ca2+ and Mn2+ had similar effects on the fluorescence of ANS with F1 and its beta subunit. The Mg2+-induced fluorescence enhancement (delta F) was high at an alkaline pH and was lowered by addition of ethylenediaminetetraacetic acid. Dicyclohexylcarbodiimide and azide had no effect on the delta F. Binding analysis showed that the concentration dependence of Mg2+ on the fluorescence enhancement of the beta subunit is similar to that of F1. These results suggest that both the beta subunit and F1 have binding sites for Mg2+ and that the delta F observed with F1 may be due to the binding of Mg2+ to the beta subunit.     

Structure of bovine mitochondrial F(1)-ATPase inhibited by Mg(2+) ADP and aluminium fluoride

BACKGROUND: The globular domain of the membrane-associated F(1)F(o)-ATP synthase complex can be detached intact as a water-soluble fragment known as F(1)-ATPase. It consists of five different subunits, alpha, beta, gamma, delta and epsilon, assembled with the stoichiometry 3:3:1:1:1. In the crystal structure of bovine F(1)-ATPase determined previously at 2.8 A resolution, the three catalytic beta subunits and the three noncatalytic alpha subunits are arranged alternately around a central alpha-helical coiled coil in the gamma subunit. In the crystals, the catalytic sites have different nucleotide occupancies. One contains the triphosphate form of the nucleotide, the second contains the diphosphate, and the third is unoccupied. Fluoroaluminate complexes have been shown to mimic the transition state in several ATP and GTP hydrolases. In order to understand more about its catalytic mechanism, F(1)-ATPase was inhibited with Mg(2+)ADP and aluminium fluoride and the structure of the inhibited complex was determined by X-ray crystallography. RESULTS: The structure of bovine F(1)-ATPase inhibited with Mg(2+)ADP and aluminium fluoride determined at 2.5 A resolution differs little from the original structure with bound AMP-PNP and ADP. The nucleotide occupancies of the alpha and beta subunits are unchanged except that both aluminium trifluoride and Mg(2+)ADP are bound in the nucleotide-binding site of the beta(DP) subunit. The presence of aluminium fluoride is accompanied by only minor adjustments in the surrounding protein. CONCLUSIONS: The structure appears to mimic a possible transition state. The coordination of the aluminofluoride group has many features in common with other aluminofluoride-NTP hydrolase complexes. Apparently, once nucleotide is bound to the catalytic beta subunit, no additional major structural changes are required for catalysis to occur.     

Substitution of betaGlu(201) in the alpha(3)beta(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 increases the affinity of catalytic sites for nucleotides

In the crystal structure of bovine mitochondrial F(1)-ATPase (MF(1)) (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the side chain oxygen of betaThr(163) interacts directly with Mg(2+) coordinated to 5'-adenylyl beta, gamma-imidodiphosphate or ADP bound to catalytic sites of beta subunits present in closed conformations. In the unliganded beta subunit present in an open conformation, the hydroxyl of betaThr(163) is hydrogen-bonded to the carboxylate of betaGlu(199). Substitution of betaGlu(201) (equivalent to betaGlu(199) in MF(1)) in the alpha(3)beta(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 with cysteine or valine increases the propensity to entrap inhibitory MgADP in a catalytic site during hydrolysis of 50 microM ATP. These substitutions lower K(m3) (the Michaelis constant for trisite ATP hydrolysis) relative to that of the wild type by 25- and 10-fold, respectively. Fluorescence quenching of alpha(3)(betaE201C/Y341W)(3)gamma and alpha(3)(betaY341W)(3)gamma mutant subcomplexes showed that MgATP and MgADP bind to the third catalytic site of the double mutant with 8.4- and 4.4-fold higher affinity, respectively, than to the single mutant. These comparisons support the hypothesis that the hydrogen bond observed between the side chains of betaThr(163) and betaGlu(199) in the unliganded catalytic site in the crystal structure of MF(1) stabilizes the open conformation of the catalytic site during ATP hydrolysis.     

Reactions of a fluorescent ATP analog, 2'-(5-dimethyl-aminonaphthalene-1-sulfonyl) amino-2'-deoxyATP, with E. coli F1-ATPase and its subunits: the roles of the high affinity binding site in the alpha subunit and the low affinity binding site in the beta subunit

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 microM. Upon binding, the fluorescence intensity of DNS-ATP at 520 nm increased exponentially with t1/2 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 alpha subunit of EF1. 4. In the presence of Mg2+, 0.65-0.82 mol of DNS-ATP binds to 1 mol of the isolated alpha subunit of EF1 with an apparent dissociation constant of 0.06-0.07 microM. Upon binding, the fluorescence intensity of DNS-ATP at 520 nm increased 1.55 fold very rapidly (t1/2 less than 1 s). No hydrolysis of DNS-ATP was observed upon the addition of the isolated alpha subunit. The fluorescence intensity of DNS-ATP was unaffected by the addition of the isolated beta subunit. DNS-ATP was also unhydrolyzed by the isolated beta subunit. 5. EF1-ATPase was reconstituted from alpha, beta, and gamma 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 alpha subunit and beta subunit, respectively. However, the findings mentioned in (4) suggest that the binding of the alpha and beta subunit, which is mediated by the gamma subunit, induces conformational change(s) in the ATP binding site located probably in the alpha subunit, and that the conformational change(s) is essential to exert the full hydrolyzing activity.     

ADP binding to TF1 and its subunits induces ultraviolet spectral changes

Adenine nucleotide binding sites on the coupling factor ATPase of thermophilic bacterium PS3 (TF1) were investigated by UV spectroscopy and by equilibrium dialysis. When ADP was mixed with TF1 in the presence and in the absence of Mg2+, an UV absorbance change was induced (t1/2 approximately 1 min) with a peak at about 278 nm and a trough at about 250 nm. Similar spectral changes were induced by ADP with the isolated beta subunits in the presence and in the absence of Mg2+, and with the isolated alpha subunits in the presence of Mg2+ although the magnitudes of the changes were different. From equilibrium dialysis measurement we identified two classes of nucleotide binding sites in TF1 in the presence of Mg2+, three high-affinity sites (Kd = 61 nM) and three low-affinity sites (Kd = 87 microM). In the absence of Mg2+, TF1 has one high-affinity site (Kd less than 10 nM) and five low-affinity sites (Kd = 100 microM). Moreover, we found a single Mg2+-dependent ADP binding site on the isolated alpha subunit and a single Mg2+-independent ADP binding site on the isolated beta subunit. From the above observations, we concluded that the three Mg2+-dependent high-affinity sites for ADP are located on the alpha subunit in TF1 and that the single high-affinity site is located on one of the beta subunits in TF1 in the absence of Mg2+.     

Oxidation of the alpha(3)(betaD311C/R333C)(3)gamma subcomplex of the thermophilic Bacillus PS3 F(1)-ATPase indicates that only two beta subunits can exist in the closed conformation simultaneously.

In the crystal structure of the bovine heart mitochondrial F(1)-ATPase (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the two liganded beta subunits, one with MgAMP-PNP bound to the catalytic site (beta(T)) and the other with MgADP bound (beta(D)) have closed conformations. The empty beta subunit (beta(E)) has an open conformation. In beta(T) and beta(D), the distance between the carboxylate of beta-Asp(315) and the guanidinium of beta-Arg(337) is 3.0-4.0 A. These side chains are at least 10 A apart in beta(E). The alpha(3)(betaD311C/R333C)(3)gamma subcomplex of TF(1) with the corresponding residues substituted with cysteine has very low ATPase activity unless it is reduced prior to assay or assayed in the presence of dithiothreitol. The reduced subcomplex hydrolyzes ATP at 50% the rate of wild-type and is rapidly inactivated by oxidation by CuCl(2) with or without magnesium nucleotides bound to catalytic sites. Titration of the subcomplex with iodo[(14)C]acetamide after prolonged treatment with CuCl(2) in the presence or absence of 1 mM MgADP revealed nearly two free sulfhydryl groups/mol of enzyme. Therefore, one pair of introduced cysteines is located on a beta subunit that exists in the open or partially open conformation even when catalytic sites are saturated with MgADP. Since V(max) of ATP hydrolysis is attained when three catalytic sites of F(1) are saturated, the catalytic site that binds ATP must be closing as the catalytic site that releases products is opening.     

Investigation of the substrate structure and metal cofactor requirements of the rat liver mitochondrial ATP synthase/ATPase complex

The F1 moiety of the rat liver mitochondrial ATP synthase/ATPase complex contains as isolated 2 mol Mg2+/mol F1, 1 mol of which is nonexchangeable and the other which is exchangeable (N. Williams, J. Hullihen, and P.L. Pedersen, (1987) Biochemistry 26, 162-169). In addition, the enzyme binds 1 mol ADP/mol F1 and 3 mol AMP.PNP, the latter of which can bind in complex formation with divalent cation and displace the Mg2+ at the exchangeable site. Thus, in terms of ligand binding sites the fully loaded rat liver F1 complex contains 3 mol MgAMP.PNP, 1 mol ADP, and 1 mol Mg2+. In this study we have used several metal ATP complexes or analogs thereof to gain further insight into the ligand binding domains of rat liver F1 and the mechanism by which it catalyzes ATP hydrolysis in soluble and membrane bound form. Studies with LaATP confirmed that MgATP is the most likely substrate for rat liver F1, and provided evidence that the enzyme may contain additional Mg2+ binding sites, undetected in previous studies of F1-ATPases, that are required for catalytic activity. Thus, F1 containing the thermodynamically stable LaATP complex in place of MgATP requires added Mg2+ to induce ATP hydrolysis. As Mg2+ cannot readily displace La2+ under these conditions there appears to be a catalytically important class of Mg2+ binding sites on rat liver F1, distinct from the nonexchangeable Mg2+ site and the sites involved in binding MgATP. Additional studies carried out with exchange inert metal-nucleotide complexes involving rhodium and the Mg2+ and Cd2+ complexes of ATP beta S and ATP alpha S imply that the rate-limiting step in the ATPase reaction pathway occurs subsequent to the P gamma-O-P beta bond cleavage steps, perhaps at the level of Mg(ADP)(Pi) hydrolysis or MgADP release. Evidence is presented that Mg2+ remains coordinated to the leaving group of the reaction, i.e., the beta phosphoryl group. Finally, in contrast to soluble F1, F1 bound to F0 in the inner mitochondrial membrane failed to discriminate between the Mg2+ complexes of the ATP beta S isomers. This indicates that a fundamental difference may exist between the catalytic or kinetic mechanism of F1 and the more physiologically intact F0F1 complex.     

Reversible inhibition of (Na+, K+) ATPase by Mg2+, adenosine triphosphate, and K+.

Adenosine triphosphate (ATP) hydrolysis catalyzed by the plasma membrane (Na+,K+)ATPase isolated from several sources was inhibited by Mg+, provided that K+ and ATP were also present. Phosphorylation of the adenosine triphosphatase (ATPase) by ATP and by inorganic phosphate was also inhibited, as was p-nitrophenyl phosphatase activity. (Ethylenedinitrilo)tetraacetic acid (EDTA) and catecholamines protected from and reversed the inhibition of ATP hydrolysis by Mg2+, K+ and ATP. EDTA was protected by chelation of Mg2+ but catecholamines acted by some other mechanism. The specificities of various nucleotides as inhibitors (in conjunction with Mg2+ and K+) and as substrates for the (Na+, K+) ATPase were strikingly different. ATP, ADP, beta,gamma-CH2-ATP and alpha,beta-CH2-ADP were active as inhibitors, whereas inosine, cytidine, uridine, and guanosine triphosphates (ITP, CTP, UTP, and GTP) and adenosine monophosphate (AMP) were not. On the other hand, ATP and CTP were substrates and beta,gamma-NH-ATP was a competitive inhibitor of ATP hydrolysis, but not an inhibitor in conjunction with Mg2+ and K+. The Ca2+-ATPase from sarcoplasmic reticulum and F1, the Mg2+-ATPase from the inner mitochondrial membrane, were also inhibited by Mg2+. Catecholamines reversed inhibition of the Ca2+-ATPase, but not that of F1.     

Cross-linking of two beta subunits in the closed conformation in F1-ATPase

In the crystal structure of mitochondrial F1-ATPase, two beta subunits with a bound Mg-nucleotide are in "closed" conformations, whereas the third beta subunit without bound nucleotide is in an "open" conformation. In this "CCO" (beta-closed beta-closed beta-open) conformational state, Ile-390s of the two closed beta subunits, even though they are separated by an intervening alpha subunit, have a direct contact. We replaced the equivalent Ile of the alpha3beta3gamma subcomplex of thermophilic F1-ATPase with Cys and observed the formation of the beta-beta cross-link through a disulfide bond. The analysis of conditions required for the cross-link formation indicates that: (i) F1-ATPase takes the CCO conformation when two catalytic sites are filled with Mg-nucleotide, (ii) intermediate(s) with the CCO conformation are generated during catalytic cycle, (iii) the Mg-ADP inhibited form is in the CCO conformation, and (iv) F1-ATPase dwells in conformational state(s) other than CCO when only one (or none) of catalytic sites is filled by Mg-nucleotide or when catalytic sites are filled by Mg2+-free nucleotide. The alpha3beta3gamma subcomplex containing the beta-beta cross-link retained the activity of uni-site catalysis but lost that of multiple catalytic turnover, suggesting that open-closed transition of beta subunits is required for the rotation of gamma subunit but not for hydrolysis of a single ATP.     

Directed mutagenesis of the beta-subunit of F1-ATPase from Escherichia coli

Oligonucleotide-directed mutagenesis was used to generate six mutant strains of Escherichia coli which had the following specific amino acid substitutions in the beta-subunit of F1-ATPase: (i) Lys-155----Gln; (ii) Lys-155----Glu; (iii) Gly-149----Ile; (iv) Gly-154----Ile; (v) Tyr-297----Phe;(vi) Tyr-354----Phe. The effects of each mutation on growth of cells on succinate plates or limiting (3 mM) glucose and on cell membrane ATPase activity and ATP-driven pH gradient formation were studied. The results showed Lys-155 to be essential for catalysis, as has been predicted previously from sequence homology and structural considerations; however, the results appear to contradict the hypothesis that Lys-155 interacts with one of the substrate phosphate groups because the Lys-155----Glu mutation was less detrimental than Lys-155----Gln. Gly-149 and Gly-154 have been predicted to be involved in essential conformational changes in F1-ATPase by virtue of their position in a putative glycine-rich flexible loop structure. The mutation of Gly-154----Ile caused strong impairment of catalysis, but the Gly-149----Ile mutation produced only moderate impairment. The two tyrosine residues chosen for mutation were residues which have previously received much attention due to their being the sites of reaction of the inactivating chemical modification reagents 4-chloro-7-nitrobenzofurazan (Tyr-297) and p-fluorosulfonylbenzoyl-5'-adenosine (Tyr-354). We found that mutation of Tyr-297----Phe caused only minor impairment of catalysis, and mutation of Tyr-354----Phe produced no impairment. Therefore, a direct role for either of these tyrosine residues in catalysis is unlikely.     

Recent developments on structural and functional aspects of the F1 sector of H+-linked ATPases

This review concerns the catalytic sector of F1 factor of the H+-dependent ATPases in mitochondria (MF1), bacteria (BF1) and chloroplasts (CF1). The three types of F1 have many similarities with respect to the structural parameters, subunit composition and catalytic mechanism. An alpha 3 beta 3 gamma delta epsilon stoichiometry is now accepted for MF1 and BF1; the alpha 2 beta 2 gamma 2 delta 2 epsilon 2 stoichiometry for CF1 remains as matter of debate. The major subunits alpha, beta and gamma are equivalent in MF1, BF1 and CF1; this is not the case for the minor subunits delta and epsilon. The delta subunit of MF1 corresponds to the epsilon subunit of BF1 and CF1, whereas the mitochondrial subunit equivalent to the delta subunit of BF1 and CF1 is probably the oligomycin sensitivity conferring protein (OSCP). The alpha beta gamma assembly is endowed with ATPase activity, beta being considered as the catalytic subunit and gamma as a proton gate. On the other hand, the delta and epsilon subunits of BF1 and CF1 most probably act as links between the F1 and F0 sectors of the ATPase complex. The natural mitochondrial ATPase inhibitor, which is a separate protein loosely attached to MF1, could have its counterpart in the epsilon subunit of BF1 and CF1. The generally accepted view that the catalytic subunit in the different F1 species is beta comes from a number of approaches, including chemical modification, specific photolabeling and, in the case of BF1, use of mutants. The alpha subunit also plays a central role in catalysis, since structural alteration of alpha by chemical modification or mutation results in loss of activity of the whole molecule of F1. The notion that the proton motive force generated by respiration is required for conformational changes of the F1 sector of the H+-ATPase complex has gained acceptance. During the course of ATP synthesis, conversion of bound ADP and Pi into bound ATP probably requires little energy input; only the release of the F1-bound ATP would consume energy. ADP and Pi most likely bind at one catalytic site of F1, while ATP is released at another site. This mechanism, which underlines the alternating cooperativity of subunits in F1, is supported by kinetic data and also by the demonstration of partial site reactivity in inactivation experiments performed with selective chemical modifiers. One obvious advantage of the alternating site mechanism is that the released ATP cannot bind to its original site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the catalytic and noncatalytic ADP binding sites of the F1-ATPase from the thermophilic bacterium, PS3

Two classes of ADP binding sites at 20 degrees C have been characterized in the F1-ATPase from the thermophilic bacterium, PS3 (TF1). One class is comprised of three sites which saturate with [3H]ADP in less than 10 s with a Kd of 10 microM which, once filled, exchange rapidly with medium ADP. The binding of ADP to these sites is dependent on Mg2+. [3H]ADP bound to these sites is removed by repeated gel filtrations on centrifuge columns equilibrated with ADP free medium. The other class is comprised of a single site which saturates with [3H]ADP in 30 min with a Kd of 30 microM. [3H]ADP bound to this site does not exchange with medium ADP nor does it dissociate on gel filtration through centrifuge columns equilibrated with ADP free medium. Binding of [3H]ADP to this site is weaker in the presence of Mg2+ where the Kd for ADP is about 100 microM. [3H]ADP dissociated from this site when ATP plus Mg2+ was added to the complex while it remained bound in the presence of ATP alone or in the presence of ADP, Pi, or ADP plus Pi with or without added Mg2+. Significant amounts of ADP in the 1:1 TF1.ADP complex were converted to ATP in the presence of Pi, Mg2+, and 50% dimethyl sulfoxide. Enzyme-bound ATP synthesis was abolished by chemical modification of a specific glutamic acid residue by dicyclohexylcarbodiimide, but not by modification of a specific tyrosine residue with 7-chloro-4-nitrobenzofurazan. Difference circular dichroism spectra revealed that the three Mg2+ -dependent, high affinity ADP binding sites that were not stable to gel filtration were on the alpha subunits and that the single ADP binding site that was stable to gel filtration was on one of the three beta subunits. It has also been demonstrated that enzyme-bound ATP is formed when the TF0.F1 complex containing bound ADP was incubated with Pi, Mg2+, and 50% dimethyl sulfoxide.     

Inactivation of beef heart mitochondrial F1-ATPase by the 2',3'-dialdehyde derivatives of adenine nucleotides

Beef heart mitochondrial F1-ATPase was inactivated by the 2',3'-dialdehyde derivatives of ATP, ADP and AMP (oATP, oADP, oAMP). In the absence of Mg2+, inactivation resulted from the binding of 1 mol nucleotide analog per active unit of F1. The most efficient analog was oADP, followed by oAMP and oATP. Complete inactivation was correlated with the binding of about 11 mol [14C]oADP/mol F1. After correction for non-specific labeling, the number of specifically bound [14C]oADP was 2-3 mol per mol F1. By SDS-polyacrylamide gel electrophoresis, [14C]oADP was found to bind covalently mainly to the alpha and beta subunits. In the presence of Mg2+, oATP behaved as a substrate and was slowly hydrolyzed.