Thermodynamic analyses of the catalytic pathway of F1-ATPase from Escherichia coli. Implications regarding the nature of energy coupling by F1-ATPases

Thermodynamic properties of 12 different F1-ATPase enzymes were analyzed in order to gain insights into the catalytic mechanism and the nature of energy coupling to delta mu H+. The enzymes were normal soluble Escherichia coli F1, a group of nine beta-subunit mutant soluble E. coli F1 enzymes (G142S, K155Q, K155E, E181Q, E192Q, M209I, D242N, D242V, R246C), and both soluble and membrane-bound bovine heart mitochondrial F1. Unisite activity was studied by use of Gibbs free energy diagrams, difference energy diagrams, and derivation of linear free energy relationships. This allowed construction of binding energy diagrams for both the unisite ATP hydrolysis and ATP synthesis reaction pathways, which were in agreement. The binding energy diagrams showed that the step of Pi binding is a major energy-requiring step in ATP synthesis, as is the step of ATP release. It is suggested that there are two major catalytic enzyme conformations, and ATP- and an ADP-binding conformation. The effects of the mutations on the rate-limiting steps of multisite as compared to unisite activity were correlated, suggesting a direct link between the rate-limiting steps of the two types of activity. Multisite activity was analyzed by Arrhenius plots and by study of relative promotion from unisite to multisite rate. Changes in binding energy due to mutation were seen to have direct effects on multisite catalysis. From all the data, a model is derived to describe the mechanism of ATP synthesis. ATP hydrolysis, and energy coupling to delta mu H+ in F1F0-ATPases.     

Further examination of seventeen mutations in Escherichia coli F1-ATPase beta-subunit.

Seventeen mutations in beta-subunit of Escherichia coli F1-ATPase which had previously been characterized in strain AN1272 (Mu-induced mutant) were expressed in strain JP17 (beta-subunit gene deletion). Six showed unchanged behavior, namely: C137Y; G142D; G146S; G207D; Y297F; and Y354F. Five failed to assemble F1F0 correctly, namely: G149I; G154I; G149I,G154I; G223D; and P403S,G415D. Six assembled F1F0 correctly, but with membrane ATPase lower than in AN1272, namely: K155Q; K155E; E181Q; E192Q; D242N; and D242V. AN1272 was shown to unexpectedly produce a small amount of wild-type beta-subunit; F1-ATPase activities reported previously in AN1272 were referable to hybrid enzymes containing both mutant and wild-type beta-subunits. Purified F1 was obtained from K155Q; K155E; E181Q; E192Q; and D242N mutants in JP17. Vmax ATPase values were lower, and unisite catalysis rate and equilibrium constants were perturbed to greater extent, than in AN1272. However, general patterns of perturbation revealed by difference energy diagrams were similar to those seen previously, and the new data correlated well in linear free energy relationships for reaction steps of unisite catalysis. Correlation between multisite and unisite ATPase activity was seen in the new enzymes. Overall, the data give strong support to previously proposed mechanisms of unisite catalysis, steady-state catalysis, and energy coupling in F1-ATPases (Al-Shawi, M. K., Parsonage, D. and Senior, A. E. (1990) J. Biol. Chem. 265, 4402-4410). The K155Q, K155E, D242N, and E181Q mutations caused 5000-fold, 4000-fold, 1800-fold, and 700-fold decrease, respectively, in Vmax ATPase, implying possibly direct roles for these residues in catalysis. Experiments with the D242N mutant suggested a role for residue beta D242 in catalytic site Mg2+ binding.     

Catalytic sites of Escherichia coli F1-ATPase. Characterization of unisite catalysis at varied pH.

Using manual rapid-mixing procedures in which small, equal volumes of Escherichia coli F1-ATPase and [gamma-32P]ATP were combined at final concentrations of 2 and 0.2 microM, respectively (i.e., unisite catalysis conditions), it was shown that greater than or equal to 66% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi equal to 0.4 and the rate of dissociation of bound [32P]Pi equal to 3.5 x 10(-3) s-1, similar to previously published values. Azide is known to inhibit cooperative but not unisite catalysis in F1-ATPase [Noumi, T., Maeda, M., & Futai, M. (1987) FEBS Lett. 213, 381-384]. In the presence of 1 mM sodium azide, 99% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi being 0.57. These experiments demonstrated that when conditions are used which minimize cooperative catalysis, most or all of the F1 molecules bind substoichiometric ATP tightly, hydrolyze it with retention of bound ATP and Pi, and release the products slowly. The data justify the validity of previously published rate constants for unisite catalysis. Unisite catalysis in E. coli F1-ATPase was studied at varied pH from 5.5 to 9.5 using buffers devoid of phosphate. Rate constants for ATP binding/release, ATP hydrolysis/resynthesis, Pi release, and ADP binding/release were measured; the Pi binding rate constant was inferred from the delta G for ATP hydrolysis. ATP binding was pH-independent; ATP release accelerated at higher pH. The highest KaATP (4.4 x 10(9) M-1) was seen at physiological pH 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete kinetic and thermodynamic characterization of the unisite catalytic pathway of Escherichia coli F1-ATPase. Comparison with mitochondrial F1-ATPase and application to the study of mutant enzymes

A complete analysis is presented of the component rate constants of the "unisite" reaction pathway in normal Escherichia coli F1-ATPase. Gibbs free energy profiles of the unisite reaction pathway were constructed for both normal E. coli F1 and bovine-heart mitochondrial F1, and comparison indicated that E. coli F1 is an ancestral form of the mitochondrial enzyme. Similar kinetic and thermodynamic analyses of the unisite reaction pathway were done for mutant beta-Asn-242 and beta-Val-242 E. coli F1-ATPases. Both mutations affected unisite binding and hydrolysis of MgATP but had little effect on release of products or binding of MgADP. It was apparent that a primary effect of the mutations was on the interaction between the catalytic nucleotide-binding domain and the substrate MgATP. The catalytic transition state [F1-ATP]++ was the most destabilized step in the reaction sequence. Measurements of delta delta G[F1.ATP]++ and linear free energy plots for the catalytic step were consistent with the view that, in normal enzyme, residue beta-Asp-242 accepts an H-bond from the transition-state substrate in order to facilitate catalysis. Both mutations impaired positive catalytic cooperativity. This was caused by energetic destabilization of the catalytic transition state and was an indirect effect, not a direct effect on signal transmission per se between catalytic nucleotide-binding domains on beta-subunits. Therefore, impairment of unisite catalysis and of positive catalytic cooperativity appeared to be linked. This may provide a unifying explanation as to why a series of other, widely separated mis-sense mutations within the catalytic nucleotide-binding domain on F1-beta-subunit, which have been reported to affect unisite catalysis, also impair positive catalytic cooperativity. Linear free energy plots for the ATP-binding step of unisite catalysis demonstrated that beta-Asn-242 and beta-Val-242 mutant enzymes did not suffer any gross disruptive change in structure of the catalytic nucleotide-binding domain, reinforcing the view that impairment of catalysis was due to a localized effect. Such analyses confirmed that six other F1-beta-subunit mutants, previously generated and characterized in this laboratory and thought to have inhibitory side-chain substitutions in the catalytic nucleotide-binding domain, are also devoid of gross structural disruption.     

The a subunit ala-217 --> arg substitution affects catalytic activity of F(1)F(0) ATP synthase

A large number of mutations affecting the F(0) sector of Escherichia coli F(1)F(0) ATP synthase have been constructed and characterized. A subset of the missense mutations resulted in fully assembled enzyme complexes blocked in proton translocation and displaying marked decreases in ATP hydrolysis activity. The catalytic activities of one such mutant enzyme, a(ala-217-->arg), have been determined using both multisite and unisite catalysis conditions. As expected, the V(max) of the a(ala-217-->arg) enzyme was reduced under conditions of saturating substrate concentration. However, the F(0) sector amino acid substitution did not affect nucleotide occupancy of the noncatalytic sites. Moreover, the microscopic rate constants measured using unisite methods yielded no significant differences between the intact wild type F(1)F(0) ATP synthase and the a(ala-217-->arg) mutant enzyme. In general, the values for unisite activities in both preparations were very similar to numbers reported in the literature for E. coli F(1)-ATPase. The results suggest that the a(ala-217-->arg) substitution resulted in a defect in catalytic cooperativity and most likely altered the enzyme by inhibiting the rotational mechanism of F(1)F(0) ATP synthase.     

Catalytic properties of Escherichia coli F1-ATPase depleted of endogenous nucleotides.

Nucleotide-depleted Escherichia coli F1 was prepared by the procedure of Wise et al. (1983, Biochem. J. 215, 343-350). This enzyme had high rates of steady-state ATPase and GTPase activity. When "unisite" ATP hydrolysis was measured using an F1/ATP concentration ratio of 10, all of the substoichiometric ATP became bound to the high-affinity catalytic site and none became bound to noncatalytic sites. The association rate constant for ATP binding was 7 x 10(5) M-1 s-1 and the KdATP was 7.9 x 10(-10) M, as compared to values of 3.8 x 10(5) M-1 s-1 and 1.9 x 10(-10) M, respectively, in native (i.e., nucleotide-replete) F1. Rate constants for bound ATP hydrolysis, ATP resynthesis, and P(i) release, and the reaction equilibrium constant, were similar in nucleotide-depleted and native F1. Therefore, we conclude that occupancy of the noncatalytic sites is not required for formation of the high-affinity catalytic site of F1 and has no significant effect on unisite catalysis. In further experiments we looked for the occurrence of inhibitory, catalytic-site-bound MgADP in E. coli F1. Such an entity has been reported for chloroplast and mitochondrial F1. However, our experiments gave no indication for inhibitory MgADP in E. coli F1.     

The defective proton-ATPase of uncD mutants of Escherichia coli. Identification by DNA sequencing of residues in the beta-subunit which are essential for catalysis or normal assembly

Six mutant uncD alleles, affecting essential residues of the beta-subunit of Escherichia coli proton-ATPase, have been identified by intragenic complementation mapping, cloning, and DNA sequencing. Five of the mutations impair catalysis but do not cause structural perturbation of F1-ATPase. The amino acid substitutions found were as follows: uncD412, Gly-142----Ser; uncD430 and uncD431, both Arg-246----Cys; uncD478, Ser-174----Phe; and uncD484, Met-209----Ile. Kinetic characteristics of each corresponding mutant F1-ATPase are described or reviewed. In each case, the major determinant of impaired catalysis appears to be an attenuation of positive catalytic site cooperativity. Additionally, each mutation affects intrinsic properties of the catalytic site, including affinity for ATP, the ratio between unisite-bound substrate and products, and the rate of release of product inorganic phosphate under unisite ATP hydrolysis conditions. These effects are discussed in terms of a structural model of the catalytic nucleotide-binding domain of beta-subunit proposed recently (Duncan, T.M., Parsonage, D., and Senior, A.E. (1986) FEBS Lett. 208, 1-6). Each of the mutations lies within that domain. The uncD409 allele abolishes normal assembly of F1-ATPase. The amino acid substitution is Gly-214----Arg, which is suggested to affect a beta-turn connecting a beta-strand and an alpha-helix in the predicted nucleotide-binding domain of the beta-subunit.     

The defective proton-ATPase of uncD mutants of Escherichia coli. Two mutations which affect the catalytic mechanism

The catalytic characteristics of F1-ATPases from uncD412 and uncD484 mutant strains of Escherichia coli were studied in order to understand how these beta-subunit mutations cause defective catalysis. Both mutant enzymes showed reduced affinity for ATP at the first catalytic site. While uncD412 F1 was similar to normal in other aspects of single site catalysis, uncD484 F1 showed a Keq of bound reactants greatly biased toward bound substrate ATP and an abnormally fast rate of Pi release. Impairment of productive catalytic cooperativity was the major cause of the reduced steady state ("multisite") catalytic rate in both mutant enzymes. Addition of excess ATP to saturate second and/or third catalytic sites did promote ATP hydrolysis and product release at the first catalytic site of uncD412 F1, but the multisite turnover rate was significantly slower than normal. In contrast, with uncD484 F1, addition of excess ATP induced rapid release of ATP from the first catalytic site and so productive catalytic cooperativity was almost completely absent. The results show that both mutations affect properties of the catalytic site and catalytic site cooperativity and further that the relatively more severe uncD484 mutation affects a residue which acts as a determinant of the fate of bound substrate ATP during promotion of catalysis. Taken together with previous studies of uncA mutant F1-ATPases (Wise, J. G., Latchney, L. R., Ferguson, A. M., and Senior, A. E. (1984) Biochemistry 23, 1426-1432) the results indicate that catalytic site cooperativity in F1-ATPases involves concerted beta-alpha-beta intersubunit communication between catalytic sites on the beta-subunits.     

H~＋－ATPase单位点水解过程中Fo构象的变化

利用Ｈ＾＋－ＡＴＰ酶复合中的Ｆｏ的色氨酸荧光,观察了复合体中Ｆ１结合ＡＴＰ或ＡＤＰ时,Ｆｏ的荧光猝灭常数的变化结果表明Ｆ１结合ＡＴＰ或ＡＤＰ时Ｆｏ可得到不同的猝来常数,也就是Ｆｏ会产生不同的构象变化。这些结果说明了Ｈ＾＋ＡＴＰ酶合ＡＴＰ合成的过程中Ｆ１与Ｆｏ之间存在着构象之间的通信与传递。     

H~＋－ATPase单位点水解过程中Fo构象的变化

利用Ｈ＋－ＡＴＰ酶复合体（也称ＡＴＰ合成酶）中的Ｆｏ的色氨酸荧光,观察了复合体中Ｆ１结合ＡＴＰ或ＡＤＰ（酶蛋白与底物分子比为１：１）时,Ｆｏ的荧光猝灭常数的变化（用竹红菌乙作为膜区蛋白荧光的猝灭剂）结果表明Ｆ１结合ＡＴＰ或ＡＤＰ时Ｆｏ可得到不同的猝灭常数（Ｋｓｖ）,也就是Ｆｏ会产生不同的构象变化。加入二价金属离子起动ＡＴＰ水解反应结束后：ＡＴＰ＋Ｈ２Ｏ→ＡＤＰ＋Ｐｉ,这时可以在Ｆｏ观察到与ＡＤＰ加Ｍｇ２＋时相同猝灭常数Ｋｓｖ;用荧光强度随时间进程变化的实验可观察到Ｆ１水解过程中导致Ｆｏ构象变化的动力学过程。这些结果说明了Ｈ＋－ＡＴＰ酶复合体ＡＴＰ合成的过程中Ｆ１与Ｆｏ之间存在着构象之间的通信与传递。     

Effects of dimethyl sulfoxide on catalysis in Escherichia coli F1-ATPase.

(1) Dimethyl sulfoxide (DMSO) markedly inhibited the Vmax of multisite ATPase activity in Escherichia coli F1-ATPase at concentrations greater than 30% (v/v). Vmax/KM was reduced by 2 orders of magnitude in 40% (v/v) DMSO at pH 7.5, primarily due to reduction of Vmax. The inhibition was rapidly reversed on dilution into aqueous buffer. (2) KdATP at the first, high-affinity catalytic site was increased 1500-fold from 2.3 x 10(-10) to 3.4 x 10(-7) M in 40% DMSO at pH 7.5, whereas KdADP was increased 3.2-fold from 8.8 to 28 microM. This suggests that the high-affinity catalytic site presents a hydrophobic environment for ATP binding in native enzyme, that there is a significant difference between the conformation for ADP binding as opposed to ATP binding, and that the ADP-binding conformation is more hydrophilic. (3) Rate constants for hydrolysis and resynthesis of bound ATP in unisite catalysis were slowed approximately 10-fold by 40% DMSO; however, the equilibrium between bound Pi/bound ATP was little changed. The reduction in catalysis rates may well be related to the large increase in KdATP (less constrained site). (4) Significant Pi binding to E. coli F1 could not be detected either in 40% DMSO or in aqueous buffer using a centrifuge column procedure. (5) We infer, on the basis of the measured constants KaATP, K2 (hydrolysis/resynthesis of ATP), k+3 (Pi release), and KdADP and from estimates of k-3 (Pi binding) that delta G for ATP hydrolysis in 40% DMSO-containing pH 7.5 buffer is between -9.2 and -16.8 kJ/mol.     

Escherichia coli ATP synthase alpha subunit Arg-376: the catalytic site arginine does not participate in the hydrolysis/synthesis reaction but is required for promotion to the steady state

The three catalytic sites of the F(O)F(1) ATP synthase interact through a cooperative mechanism that is required for the promotion of catalysis. Replacement of the conserved alpha subunit Arg-376 in the Escherichia coli F(1) catalytic site with Ala or Lys resulted in turnover rates of ATP hydrolysis that were 2 x 10(3)-fold lower than that of the wild type. Mutant enzymes catalyzed hydrolysis at a single site with kinetics similar to that of the wild type; however, addition of excess ATP did not chase bound ATP, ADP, or Pi from the catalytic site, indicating that binding of ATP to the second and third sites failed to promote release of products from the first site. Direct monitoring of nucleotide binding in the alphaR376A and alphaR376K mutant F(1) by a tryptophan in place of betaTyr-331 (Weber et al. (1993) J. Biol. Chem. 268, 20126-20133) showed that the catalytic sites of the mutant enzymes, like the wild type, have different affinities and therefore, are structurally asymmetric. These results indicate that alphaArg-376, which is close to the beta- or gamma-phosphate group of bound ADP or ATP, respectively, does not make a significant contribution to the catalytic reaction, but coordination of the arginine to nucleotide filling the low-affinity sites is essential for promotion of rotational catalysis to steady-state turnover.     

Defective proton ATPase of uncA mutants of Escherichia coli. 5'-Adenylyl imidodiphosphate binding and ATP hydrolysis

The Escherichia coli uncA gene codes for the alpha-subunit of the F1 sector of the membrane proton ATPase. In this work purified soluble F1 enzymes from three mutant strains ( uncA401 , uncA447 , and uncA453 ) have been compared to F1 from a normal strain in respect to (a) binding of 5'-adenylyl imidodiphosphate (AMPPNP) to native enzyme in both the presence and absence of Mg, (b) high-affinity binding of MgATP to native enzyme, (c) total reloading of MgAMPPNP to nucleotide-depleted F1 preparations, (d, e) ability to hydrolyze MgATP at both high MgATP concentrations (d) (steady-state conditions) and low MgATP concentrations (e) where substrate hydrolysis occurs under nonsteady-state (" unisite ") conditions, and (f) sensitivity of steady-state ATPase activities to inhibitors of normal F1-ATPase activity. uncA mutant F1 showed normal stoichiometry of MgAMPPNP binding to both native (three sites per F1) and nucleotide-depleted preparations (six sites per F1). Native uncA F1 preparations showed lower-than-normal affinity for MgAMPPNP and MgATP at the first site filled. Binding of AMPPNP in the absence of Mg was similar to normal, except that no increase in affinity for AMPPNP was induced by aurovertin. The uncA F1-ATPases had low but real steady-state rates of ATP hydrolysis, which were inhibited by aurovertin but relatively insensitive to inhibition by AMPPNP, efrapeptin, and sodium azide. Non-steady-state ( unisite ) ATP hydrolysis rates catalyzed at low substrate concentrations by uncA F1-ATPases were similar to normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Directed mutations of the strongly conserved lysine 155 in the catalytic nucleotide-binding domain of beta-subunit of F1-ATPase from Escherichia coli

The amino acid sequence -Gly-X-X-X-X-Gly-Lys- occurs in many, diverse, nucleotide-binding proteins, and there is evidence that it forms a flexible loop which interacts with one or other of the phosphate groups of bound nucleotide. This sequence occurs as -Gly-Gly-Ala-Gly-Val-Gly-Lys- in the beta-subunit of the enzyme F1-ATPase, where it is thought to form part of the catalytic nucleotide-binding domain. Mutants of Escherichia coli were generated in which residue beta-lysine 155, at the end of the above sequence, was replaced by glutamine or glutamate. Properties of the soluble purified F1-ATPase from each mutant were studied. The results showed: 1) replacement of lysine 155 by Gln or Glu decreased the steady-state rate of ATP hydrolysis by 80 and 66%, respectively. 2) Characteristics of ATP hydrolysis at a single site were not markedly changed in the mutant enzymes, implying that lysine 155 is not directly involved in bond cleavage during ATP hydrolysis or bond formation during ATP synthesis. 3) The binding affinity for MgATP was weakened considerably in the mutants (Lys much much greater than Gln greater than Glu), whereas the binding affinity for MgADP was affected only mildly (Lys = Gln greater than Glu), suggesting that lysine 155 interacts with the gamma-phosphate of ATP bound at a single high affinity catalytic site. 4) The major determinant of inhibition of steady-state ATPase turnover rate in the mutant enzymes was an attenuation of positive catalytic cooperativity. 5) The data are consistent with the idea that during multisite catalysis residue 155 of beta-subunit undergoes conformational movement which changes substrate and product binding affinities.     

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)     

Importance of the conserved Walker B glutamate residues, 556 and 1201, for the completion of the catalytic cycle of ATP hydrolysis by human P-glycoprotein (ABCB1)

The human MDR1 (ABCB1) gene product, P-glycoprotein (Pgp), functions as an ATP-dependent efflux pump for a variety of chemotherapeutic drugs. In this study, we assessed the role of conserved glutamate residues in the Walker B domain of the two ATP sites (E556 and E1201, respectively) during the catalytic cycle of human Pgp. The mutant Pgps (E556Q, E556A, E1201Q, E1201A, E556/1201Q, and E556/1201A) were characterized using a vaccinia virus based expression system. Although steady-state ATP hydrolysis and drug transport activities were abrogated in both E556Q and E1201Q mutant Pgps, [alpha-(32)P]-8-azidoADP was trapped in the presence of vanadate (Vi), and the release of trapped [alpha-(32)P]-8-azidoADP occurred to a similar extent as in wild-type Pgp. This indicates that these mutations do not affect either the first hydrolysis event or the ADP release step. Similar results were also obtained when Glu residues were replaced with Ala (E556A and E1201A). Following the first hydrolysis event and release of [alpha-(32)P]-8-azidoADP, both E556Q and E1201Q mutant Pgps failed to undergo another cycle of Vi-induced [alpha-(32)P]-8-azidoADP trapping. Interestingly, the double mutants E556/1201Q and E556/1201A trapped [alpha-(32)P]-8-azidoADP even in the absence of Vi, and the occluded nucleotide was not released after incubation at 37 degrees C for an extended period. In addition, the properties of transition state conformation of the double mutants generated in the absence of Vi were found to be similar to that of the wild-type protein trapped in the presence of Vi (Pgp x [alpha-(32)P]-8-azidoADP xVi). Thus, in contrast to the single mutants, the double mutants appear to be defective in the ADP release step. In aggregate, these data suggest that E556 and E1201 residues in the Walker B domains may not be critical as catalytic carboxylates for the cleavage of the bond between the gamma-P and the beta-P of ATP during hydrolysis but are essential for the second ATP hydrolysis step and completion of the catalytic cycle.     

The Escherichia coli F1F0 ATP synthase displays biphasic synthesis kinetics

The F1F0 proton-translocating ATPase/synthase is the primary generator of ATP in most organisms growing aerobically. Kinetic assays of ATP synthesis have been conducted using enzymes from mitochondria and chloroplasts. However, limited data on ATP synthesis by the model Escherichia coli enzyme are available, mostly because of the lack of an efficient and reproducible assay. We have developed an optimized assay and have collected synthase kinetic data over a substrate concentration range of 2 orders of magnitude for both ADP and Pi from the synthase enzyme of E. coli. Negative and positive cooperativity of substrate binding and positive catalytic cooperativity were all observed. ATP synthesis displayed biphasic kinetics for ADP indicating that 1) the enzyme is capable of catalyzing efficient ATP synthesis when only two of three catalytic sites are occupied by ADP; and 2) occupation of the third site further activates the rate of catalysis.     

Involvement of ATP synthase residues alphaArg-376, betaArg-182, and betaLys-155 in Pi binding

alphaArg-376, betaLys-155, and betaArg-182 are catalytically important ATP synthase residues that were proposed to be involved in substrate Pi binding and subsequent steps of ATP synthesis [Senior, A.E., Nadanaciva, S. and Weber, J. (2002) Biochim. Biophys. Acta 1553, 188-211]. Here, it was shown using purified Escherichia coli F(1)-ATPase that whereas Pi protected wild-type from reaction with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, mutations betaK155Q, betaR182Q, betaR182K, and alphaR376Q abolished protection. Therefore, in ATP synthesis initial binding of substrate Pi in open catalytic site betaE is supported by each of these three residues.     

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.     

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.