The effect of dimethylsulfoxide on adenine nucleotide binding and ATP synthesis by beef-heart mitochondrial F1 ATPase.

Dimethylsulfoxide (Me2SO; 30%, v/v) promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F1 ATPase. The effects of this solvent on the adenine nucleotide binding properties of beef-heart mitochondrial F1 ATPase were examined. The ATP analog adenylyl-5'-imidodiphosphate bound to F1 at 1.9 and 1.0 sites in aqueous and Me2SO systems, respectively, with a KD value of 2.2 microM. Lower affinity sites were present also. Binding of ATP or adenylyl-5'-imidodiphosphate at levels near equimolar with the enzyme occurred to a greater extent in the absence of Me2SO. Addition of ATP to the nucleotide-loaded enzyme resulted in exchange of about one-half of the bound ATP. This occurred only in an entirely aqueous medium. ATP bound in Me2SO medium was not released by exogenous ATP. Comparison of the effect of different concentrations of Me2SO on ADP binding to F1 and ATP synthesis by the enzyme showed that binding of ADP was diminished by concentrations of Me2SO lower than those required to support ATP synthesis. However, one site could still be filled by ADP at concentrations of Me2SO optimal for ATP synthesis. This site is probably a noncatalytic site, since the nucleotide bound there was not converted to ATP in 30% Me2SO. The ATP synthesized by F1 in Me2SO originated from endogenous bound ADP. We conclude that 30% Me2SO affects the adenine nucleotide binding properties of the enzyme. The role of this in the promotion of the formation of ATP from ADP and phosphate is discussed.     

Classification of nucleotide binding sites on mitochondrial F1-ATPase from yeast

Methods are described to classify nucleotide binding sites of the mitochondrial coupling factor F₁ from yeast on the basis of their affinities and stability properties. High affinity sites or states for ATP and related adenine analogs and low affinity sites or states which bind a broad range of different nucleotide triphosphates are found. The results are discussed in terms of a two site, two cycle scheme, where binding of nucleotide at one site facilitates the release of nucleotide at a second site.     

Modulation of nucleotide binding to the catalytic sites of thermophilic F1-ATPase by the ε subunit: Implication for the role of the ε subunit in ATP synthesis

Effect of ε subunit on the nucleotide binding to the catalytic sites of F₁-ATPase from the thermophilic Bacillus PS3 (TF₁) has been tested by using α₃β₃γ and α₃β₃γε complexes of TF₁ containing βTyr341 to Trp substitution. The nucleotide binding was assessed with fluorescence quenching of the introduced Trp. The presence of the ε subunit weakened ADP binding to each catalytic site, especially to the highest affinity site. This effect was also observed when GDP or IDP was used. The ratio of the affinity of the lowest to the highest nucleotide binding sites had changed two orders of magnitude by the ε subunit. The differences may relate to the energy required for the binding change in the ATP synthesis reaction and contribute to the efficient ATP synthesis.     

Adenine nucleotide-binding sites on beef heart F1-ATPase. Conditions that affect occupancy of catalytic and noncatalytic sites

Beef heart mitochondrial F1 contains a total of six adenine nucleotide-binding sites including at least two different types of sites. Three "exchangeable" sites exchange rapidly during hydrolysis of MgATP, whereas three "nonexchangeable" sites do not (Cross, R. L. and Nalin, C. M. (1982) J. Biol. Chem. 257, 2874-2881). When F1 that has been stored as a suspension in (NH4)2SO4/ATP/EDTA/sucrose/Tris, pH 8.0, is pelleted, rinsed with (NH4)2SO4, dissolved, and desalted, it retains three bound adenine nucleotides. We find that two of these endogenous nucleotides are bound at nonexchangeable sites and one at an exchangeable site. The vacant nonexchangeable site is highly specific for adenine nucleotide and is rapidly filled by ADP upon addition of ADP or during ATP hydrolysis. ADP bound at this site can be removed by reprecipitating the enzyme with (NH4)2SO4. The single nucleotide retained by desalted F1 at an exchangeable site is displaced during hydrolysis of ATP, GTP, or ITP. The binding of PPi at two sites on the enzyme also promotes its dissociation. Neither procedure affects retention of nucleotide at the nonexchangeable sites. These observations, combined with the finding that PPi is much more easily removed from exchangeable sites than ADP, have led to the development of a procedure for preparing F1 with uniform and well-defined nucleotide site occupancy. This involves sequential exposure to MgATP, PPi, and high concentrations of Pi. Unbound ligand is removed between each step. The resulting enzyme, F1[3,0], has three occupied nonexchangeable sites and three vacant exchangeable sites. Evidence that nonexchangeable and exchangeable sites represent noncatalytic and catalytic sites, respectively, suggest that this form of the enzyme will prove useful in numerous applications, including transient kinetic measurements and affinity labeling of active site residues.     

pH dependent changes in ADP and ATP affinity for the tight nucleotide-binding site of chloroplast coupling factor 1

The pH-dependence of ADP and ATP affinity for CF₁ tight nucleotide-binding sites was studied under conditions of equilibrium between bound and free labeled nucleotides. With the nucleotide/CF₁ ratio>1, the ATP content in tightly bound nucleotides depended only slightly on medium pH. With the nucleotide/CF₁ ratio approaching 1, tightly bound ATP content grew rapidly with decreasing pH. Calculations of ADP/ATP ratio in free and tightly bound nucleotides showed that decreasing the pH from 8.0 to 6.0 induced a 150 times greater affinity of the nucleotide-binding site for ATP than for ADP. The data indicates that ATP-ADP equilibrium at the CF₁ tight nucleotide-binding site depends on protonation of specific acid-base groups of the enzyme.Abbreviations CF₁, BF₁, and MF₁ coupling factors of chloroplasts, bacteria, and mitochondria, respectively - AdN adenine nucleotide     

ADENINE NUCLEOTIDE-INDUCED CONTRACTION OF THE INNER MITOCHONDRIAL MEMBRANE : II. Effect of Bongkrekic Acid

In bovine heart mitochondria bongkrekic acid at concentrations as low as about 4 nmol/mg protein (a) completely inhibits phosphorylation of exogenous adenosine diphosphate (ADP) and dephosphorylation of exogenous adenosine triphosphate (ATP), (b) completely reverses atractyloside inhibition of inner membrane contraction induced by exogenous adenine nucleotides, and (c) decreases the amount of adenine nucleotide required to elicit maximal exogenous adenine nucleotide-induced inner membrane contraction to a level which appears to correspond closely with the concentration of contractile, exogenous adenine nucleotide binding sites Bongkrekic acid at concentrations greater than 4 nmol/mg protein induces inner membrane contraction which seems to depend on the presence of endogenous ADP and/or ATP. The findings appear to be consistent with the interpretations (a) that the inner mitochondrial membrane contains two types of contractile, adenine nucleotide binding sites, (b) that the two sites differ markedly with regard to adenine nucleotide affinity, (c) that the high affinity site is identical with the adenine nucleotide exchange carrier, (d) that the low affinity site is accessible exclusively to endogenous adenine nucleotides and is largely unoccupied in the absence of bongkrekic acid, and (e) that bongkrekic acid increases the affinity of both sites in proportion to the amount of the antibiotic bound to the inner membrane.     

Properties of Bound Inorganic Phosphate on Bovine Mitochondrial F1F0-ATP Synthase

Beef-heart mitochondrial F₁F₀-ATP synthase contained six molecules of bound inorganic phosphate (P_i). This phosphate exchanged completely with exogenous ³²P_i when the enzyme was exposed to 30% (v/v) dimethyl sulfoxide (DMSO) and then returned to a DMSO-free buffer (Beharry and Bragg 2001). Only two molecules were replaced by ³²P_i when the enzyme was not pretreated with DMSO. These two molecules of ³²P_i were not displaced from the enzyme by the treatment with 1 mM ATP. Similarly, two molecules of bound ³²P_i remained on the DMSO-pretreated enzyme following addition of ATP, that is, four molecules of ³²P_i were displaced by ATP. The ATP-resistant ³²P_i was removed from the enzyme by pyrophosphate. It is proposed that these molecules of ³²P_i are bound at an unfilled adenine nucleotide-binding noncatalytic site on the enzyme. Brief exposure of the enzyme loaded with two molecules of ³²P_i to DMSO, followed by removal of the DMSO, resulted in the loss of the bound ³²P_i and in the formation of two molecules of bound ATP from exogenous ADP. A third catalytic site on the enzyme was occupied by ATP, which could undergo a P_i ATP exchange reaction with bound P_i The presence of two catalytic sites containing bound P_i is consistent with the X-ray crystallographic structure of F₁ (Bianchet, et al., 1998). Thus, five of the six molecules of bound P_i were accounted for. Three molecules of bound P_i were at catalytic sites and participated in ATP synthesis or P_i ATP exchange. Two other molecules of bound P_i were present at a noncatalytic adenine nucleotide-binding site. The location and role of the remaining molecule of bound P_i remains to be established. We were unable to demonstrate, using chemical modification of sulfhydryl groups by iodoacetic acid, any gross difference in the conformation of F₁F₀ in DMSO-containing compared with DMSO-free buffers.     

Interaction of homogeneous mitochondrial ATPase from rat liver with adenine nucleotides and inorganic phosphate

Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, K_D = 1–2 μM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (K_I = 240–300 μM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This “tightly bound” ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetic studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, P_i can maintain the active form of the enzyme.     

Effect of the natural ATPase inhibitor on the binding of adenine nucleotides and inorganic phosphate to mitochondrial F1-ATPase

(1) Incubation of the beef heart mitochondrial ATPase, F₁ with Mg-ATP was required for the binding of the natural inhibitor, IF₁, to F₁ to form the inactive F₁-IF₁ complex. When F₁ was incubated in the presence of [¹⁴C]ATP and MgCl₂, about 2 mol ¹⁴C-labeled adenine nucleotides were found to bind per mol of F₁; the bound ¹⁴C-labeled nucleotides consisted of [¹⁴C]ADP arising from [¹⁴C]ATP hydrolysis and [¹⁴C]ATP. The ¹⁴C-labeled nucleotide binding was not prevented by IF₁. These data are in agreement with the idea that the formation of the F₁-IF₁ complex requires an appropriate conformation of F₁. (2) The ¹⁴C-labeled adenine nucleotides bound to F₁ following preincubation of F₁ with Mg-[¹⁴C]ATP could be exchanged with added [³H]ADP or [³H]ATP. No exchange occurred between added [³H]ADP or [³H]ATP and the ¹⁴C-labeled adenine nucleotides bound to the F₁-IF₁ complex. These data suggest that the conformation of F₁ in the isolated F₁-IF₁ complex is further modified in such a way that the bound ¹⁴C-labeled nucleotides are no longer available for exchange. (3) ³²P_i was able to bind to isolated F₁ with a stoichiometry of about 1 mol of P_i per mol of F₁ (Penefsky, H.S. (1977) J. Biol. Chem. 252, 2891–2899). There was no binding of ³²P_i to the F₁-IF₁ complex. Thus, not only the nucleotides sites, but also the P_i site, are masked from interaction with external ligands in the isolated F₁-IF₁ complex.     

Properties of noncatalytic sites of thioredoxin-activated chloroplast coupling factor 1

Nucleotide binding properties of two vacant noncatalytic sites of thioredoxin-activated chloroplast coupling factor 1 (CF₁) were studied. Kinetics of nucleotide binding to noncatalytic sites is described by the first-order equation that allows for two nucleotide binding sites that differ in kinetic features. Dependence of the nucleotide binding rate on nucleotide concentration suggests that tight nucleotide binding is preceded by rapid reversible binding of nucleotides. ADP binding is cooperative. The preincubation of CF₁ with Mg²⁺ produces only slight effect on the rate of ADP binding and decreases the ATP binding rate. The ATP and ADP dissociation from noncatalytic sites is described by the first-order equation for similar sites with dissociation rate constants k₋₂(ADP)=1.5×10⁻¹ min⁻¹ and k₋₂(ATP)≅10⁻³ min⁻¹, respectively. As follows from the study, the noncatalytic sites of CF₁ are not homogeneous. One of them retains the major part of endogenous ADP after CF₁ precipitation with ammonium sulfate. Its other two sites can bind both ADP and ATP but have different kinetic parameters and different affinity for nucleotides.     

Tightly-bound ATP and ADP in reconstituted submitochondrial particles.

Soluble beef-heart mitochondrial ATPase (F₁) which was depleted of tightly bound nucleotide was reconstituted with depleted submitochondrial particles and oligomycin-sensitivity conferring protein. A correlation was noted between the recovery of energy-transduction capability and the reloading of tightly bound nucleotide. Reconstituted membrane-bound F₁ contained both ATP and ADP tightly bound; the total (ATP and ADP) was tentatively calculated to be around 3.6 moles per mole membrane-bound F₁.     

ADP and ATP binding to noncatalytic sites of thiol-modulated chloroplast ATP synthase

A modified ‘cold chase’ technique was used to study tight [¹⁴C]ADP and [¹⁴C]ATP binding to noncatalytic sites of chloroplast ATP synthase (CF₀F₁). The binding was very low in the dark and sharply increased with light intensity. Dissociation of labeled nucleotides incorporated into noncatalytic sites of CF₀F₁ or CF₁ reconstituted with EDTA-treated thylakoid membranes was also found to be light-dependent. Time dependence of nucleotide dissociation is described by the first order equation with a k _d of about 5 min⁻¹. The exposure of thylakoid membranes to 0.7–24.8 μM nucleotides leads to filling of up to two noncatalytic sites of CF₀F₁. The sites differ in their specificity: one preferentially binds ADP, whereas the other – ATP. A much higher ATP/ADP ratio of nucleotides bound at noncatalytic sites of isolated CF₁ dramatically decreases upon its reconstitution with EDTA-treated thylakoid membranes. It is suggested that the decrease is caused by conformational changes in one of the α subunits induced by its interaction with the δ subunit and/or subunit I–II when CF₁ becomes bound to a thylakoid membrane.     

Relationships of inosine triphosphate and bicarbonate effects on F1 ATPase to the binding change mechanism

Two interesting previously reported properties of mitochondrial F₁ ATPase have been confirmed and have been examined by¹⁸O exchange measurements to assess if they are consistent with sequential participation of catalytic sites during ATP hydrolysis. These are the ability of HCO ₃ ^– to increase reaction rate with apparent loss of cooperative interaction between subunits and the ability of ITP to accelerate the hydrolysis of a low concentration of ATP. The effect of HCO ₃ ^– was tested at concentrations of ATP lower than previous measurements. The activation disappeared when ATP was reduced to 0.1 µM. The HCO ₃ ^– activation at higher ATP concentrations did not change the extent of reversal of the cleavage of tightly bound ATP at the catalytic site, as measured by the average number of water oxygens incorporated with each P_i formed when 5 or 10 µM ATP is hydrolyzed. The data are consistent with sequential site participation with HCO ₃ ^– acceleration of ADP departure after a binding change that stops¹⁸O exchange and loosens ADP binding.When ITP concentration was lowered during net ITP hydrolysis by F₁ ATPase an increase in water oxygen incorporation into P_i formed is observed, as noted previously for ATP hydrolysis. The acceleration of the cleavage of a constant low concentration of [-¹⁸O]ATP by concomitant hydrolysis of increasing concentrations of ITP was accompanied by a decrease in water oxygen incorporation with each P_i formed from the ATP. These results add to evidence for the binding change mechanism for F₁ ATPase with sequential participation of catalytic sites.     

Changes in the adenine nucleotide content of beef-heart mitochondrial F1 ATPase during ATP synthesis in dimethyl sulfoxide.

Beef-heart mitochondrial F1 ATPase can be induced to synthesize ATP from ADP and inorganic phosphate in 30% Me2SO. We have analyzed the adenine nucleotide content of the F1 ATPase during the time-course of ATP synthesis, in the absence of added medium nucleotide, and in the absence and presence of 10 mM inorganic phosphate. The enzyme used in these investigations was either pretreated or not pretreated with ATP to produce F1 with a defined nucleotide content and catalytic or noncatalytic nucleotide-binding site occupancy. We show that the mechanism of ATP synthesis in Me2SO involves (i) an initial rapid loss of bound nucleotide(s), this process being strongly influenced by inorganic phosphate; (ii) a rebinding of lost nucleotide; and (iii) synthesis of ATP from bound ADP and inorganic phosphate.     

Adenine nucleotides as allosteric effectors of pea seed glutamine synthetase

The effects of adenine nucleotides on pea seed glutamine synthetase (EC 6.3.1.2) activity were examined as a part of our investigation of the regulation of this octameric plant enzyme. Saturation curves for glutamine synthetase activity versus ATP with ADP as the changing fixed inhibitor were not hyperbolic; greater apparent Vmax values were observed in the presence of added ADP than the Vmax observed in the absence of ADP. Hill plots of data with ADP present curved upward and crossed the plot with no added ADP. The stoichiometry of adenine nucleotide binding to glutamine synthetase was examined. Two molecules of [gamma-32P]ATP were bound per subunit in the presence of methionine sulfoximine. These ATP molecules were bound at an allosteric site and at the active site. One molecule of either [gamma-32P]ATP or [14C]ADP bound per subunit in the absence of methionine sulfoximine; this nucleotide was bound at an allosteric site. ADP and ATP compete for binding at the allosteric site, although ADP was preferred. ADP binding to the allosteric site proceeded in two kinetic phases. A Vmax value of 1.55 units/mg was measured for glutamine synthetase with one ADP tightly bound per enzyme subunit; a Vmax value of 0.8 unit/mg was measured for enzyme with no adenine nucleotide bound at the allosteric site. The enzyme activation caused by the binding of ADP to the allosteric sites was preceded by a lag phase, the length of which was dependent on the ADP concentration. Enzyme incubated in 10 mM ADP bound approximately 4 mol of ADP/mol of native enzyme before activation was observed; the activation was complete when 7-8 mol of ADP were bound per mol of the octameric, native enzyme. The Km for ATP (2 mM) was not changed by ADP binding to the allosteric sites. ADP was a simple competitive inhibitor (Ki = 0.05 mM) of ATP for glutamine synthetase with eight molecules of ADP tightly bound to the allosteric sites of the octamer. Binding of ATP to the allosteric sites led to marked inhibition.     

Effect of Methanol on the Nucleotide Binding to High-Affinity Sites on Chloroplast Coupling Factor 1

The effects of methanol on the nucleotide binding to isolatedchloroplast coupling factor 1 (CF₁) were investigated. IsolatedCF₁ has four kinds of nucleotide binding sites; a barely dissociableADP-binding site (site A), two slowly exchangeable high-affinitysites with different affinities for ADP (sites B and C) whichare not catalytic sites, and several low-affinity sites (Hisaboriand Sakurai 1984). Methanol at 20% (v/v) slightly acceleratedthe binding of ADP to CF₁ but did not influence the number ofbinding sites. Methanol at 10–24% (v/v) affected neitherthe total amounts of bound adenine nucleotides (2.5 mol/molCF₁) nor the incorporation of labeled ADP from the medium (1.5mol/mol CF₁ into the slowly exchangeable sites (sites A, B,C). These results indicate that no appreciable exchange of ADPoccurred at site A at 10–24% (v/v) methanol and excludethe possibility of direct participation of nucleotide bindingat this site in the regulation of ATPase. In 32% methanol, theamount of the labeled ADP bound increased, suggesting some exchangeat site A. Methanol at 20% (v/v) greatly increased the affinitiesof sites B and C for ADP, CDP, GDP, UDP and PP_i. Conformational change of CF₁ induced by the binding of nucleotidesto site(s) B (and C) increased the resistance of CF₁ to inactivationby methanol at high concentrations or by cold treatment. (Received August 16, 1984; Accepted January 23, 1985)     

Interaction of ox heart mitochondrial F1-ATPase with immobilized ADP and ATP.

The reaction of mitochondrial F1-ATPase with immobilized substrate was studied by using columns of agarose-hexane-ATP. Mg2+ was required for binding of the enzyme to the column matrix. The column-bound enzyme could be eluted fully by ATP and other nucleoside triphosphates. Nucleoside di- and mono-phosphates were less effective. At a fixed concentration of nucleotide the effectiveness of elution was proportional to the charge on the eluting molecule. The ATP of the column matrix was hydrolysed by the bound F1-ATPase to release phosphate, probably by a uni-site reaction mechanism. Thus the F1-ATPase was bound to the immobilized ATP by a catalytic site. Treatment of the bound F1-ATPase with 4-chloro-7-nitrobenzofurazan prevented complete release of the enzyme by ATP. Only one-third of the bound enzyme was now eluted by the nucleotide. The inhibition of release could be due either to the inhibitor blocking co-operative interactions between sites or to its increasing the tightness of binding of immobilized ADP at the catalytic site.     

Characterization of six nucleotide-binding sites on chloroplast coupling factor 1 and one site on its purified beta subunit

By using gel filtration chromatography, following the technique of Hummel and Dreyer (Hummel, J., and Dreyer, W. (1962) Biochim. Biophys. Acta 63, 532-534), the adenine nucleotide-binding sites of isolated soluble chloroplast ATPase (CF1) and of the beta subunit were studied. CF1 possesses six adenine nucleotide-binding sites: two high affinity sites for ADP or ATP (KdH = 1-5 microM) in addition to one site where endogenous not-exchangeable ADP is bound, and three low affinity sites binding ADP or ATP with a dissociation constant (KdL = 15-20 microM) which is considerably increased in the presence of pyrophosphate. KdH is not modified by addition of pyrophosphate. The stability of nucleotide binding at the low affinity sites increases after heat activation of CF1. Removal of the delta or epsilon subunits on CF1 affects neither the number nor the binding parameters of the nucleotide-binding sites. The purified beta subunit possesses one easily exchangeable site/subunit. It is proposed that the low affinity sites on CF1 are the catalytic sites.     

Adenine nucleotide binding sites in normal and mutant adenosine triphosphatases of Escherichia coli

Three types of assays were used to characterize adenine nucleotide binding sites on the Ca²⁺, Mg²⁺-activated ATPase of normal Escherichia coli and its unc A 401 and unc D 412 mutants. ADP was bound mainly at a single site in normal and mutant ATPase. In the absence of divalent cations ATP was bound at a single high-affinity and three low-affinity sites in normal and unc D ATPases. The 2′,3′-dialdehyde (oADP) obtained by periodate oxidation of ADP reacted with both low- and high-affinity sites whereas oATP was bound primarily at a low-affinity site. Two types of adenine nucleotide binding sites, a high-affinity site reacting with ATP and ADP and a low-affinity site for ATP, were detected by the effects of these nucleotides on the fluorescence of the aurovertin D-ATPase complex. This high-affinity site(s) was present in normal and mutant ATPases. However, the fluorescence response at both high- and low-affinity sites was modified in the unc D ATPase as a consequence of the abnormal β subunit in this enzyme. Normal fluorescence responses were not induced by the binding of oADP or oATP to the ATPases. ATP was bound at a single site on isolated α subunits of the enzyme. Since this site was not detected in the unc A ATPase, it is unlikely to be the high-affinity site detected in the intact enzyme or the binding site for the endogenous tightly bound adenine nucleotides found in the purified ATPase. It is more probable that the site detected on the isolated α subunit from the normal enzyme is that which binds oADP since this site was absent in the unc A ATPase. Pretreatment of the normal ATPase with either N, N′-dicyclohexyl-carbodiimide (DCCD) or with 4-chloro-7-nitrobenzofurazan (NbfCl), reagents which inhibit ATPase activity by reacting with a β subunit, affected binding of oADP to α subunit(s) but had less effect with oATP. Inhibition of oADP binding could be due to conformational changes induced in the α subunit by the reaction of DCCD and NbfCl with a β subunit, or to steric reasons. If the latter hypothesis is correct, the active site of the ATPase would be at the interface between α and β subunits of the enzyme.     

Pyridoxylation of essential lysine residues of mitochondrial adenosine triphosphatase

1. Soluble beef-heart mitochondrial ATPase (F₁) was incubated with [³H]pyridoxal 5′-phosphate and the Schiffbase complex formed was reduced with sodium borohydride. Spectral measurements indicate that lysine residues are modified and gel electrophoresis in the presence of detergent shows the tritium label to be associated with the two largest subunits, α and β. 2. In the absence of protecting ligands, the loss of ATP hydrolysis activity is linearly dependent on the level of pyridoxylation with complete inactivation correlating to 10 mol pyridoxamine phosphate incorporated per mol enzyme. Partial inactivation of F₁ with pyridoxal phosphate has no effect on either the K_m for ATP or the ability of bicarbonate to stimulate residual hydrolysis activity, suggesting a mixed population of fully active and fully inactive enzyme. 3. In the presence of excess magnesium, the addition of ADP or ATP, but not AMP, decreases the rate and extent of modification of F₁ by pyridoxal phosphate. The non-hydrolyzable ATP analog, 5′-adenylyl-β, γ-imidodiphosphate, is particularly effective in protecting F₁ against both modification and inactivation. Efrapeptin and P_i have no effect on the modification reaction. 4. Prior modification of F₁ with pyridoxal phosphate decreases the number of exchangeable nucleotide binding sites by one. However, pyridoxylation of F₁ is ineffective in displacing endogenous nucleotides bound at non-catalytic sites and does not affect the stoichiometry of P_i binding. 5. The ability of nucleotides to protect against modification and inactivation by pyridoxal phosphate and the loss of one exchangeable nucleotide site with the pyridoxylation of F₁ suggest the presence of a positively charged lysine residue at the catalytic site of an enzyme that binds two negatively charged substrates.