Beef Heart Mitochondrial ATPase Research Articles

Energetics of ATP Dissociation from the Mitochondrial ATPase during Oxidative Phosphorylation

The dissociation constant (KdATP) for ATP bound in the high affinity catalytic site of membrane-bound beef heart mitochondrial ATPase (F1) was calculated from the ratio of the rate constants for the reverse dissociation step (k-1) and the forward binding step (k+1). k-1 for ATP bound to submitochondrial particles or to submitochondrial particles washed with KCl so as to activate ATPase activity was accelerated by about five orders of magnitude during respiratory chain-linked oxidations of NADH. In the presence of NADH and 0.1 mM ADP, k-1 increased more than six orders of magnitude. These energy-dependent dissociations of ATP were sensitive to the uncoupler carbonyl cyanide p-trifluoromethyloxyphenylhydrazone. Only small changes in k+1 were observed in the presence of NADH or NADH and ADP. KdATP at 23 degrees C in the absence of NADH and ADP was 10(-12) M, in the presence of NADH, 3 microM, and in the presence of NADH and 0.1 mM ADP, 60 microM. Thus, the dissociation of ATP during the transition from non-energized to energized states was, under these conditions, accompanied by observed free energy changes of 8 and 9.7 kcal/mol, respectively.

Covalent modification of catalytic sites on membrane-bound beef heart mitochondrial ATPase by 2-azido-adenine nucleotides.

Incubation in the dark of 32P-labeled 2-azido-adenine nucleotides with submitochondrial particles from beef heart led to tight binding of the label by membrane-bound F1. That is, the label remained with the particles following two passages through centrifuge columns. After removal of free nucleotides and ultraviolet irradiation, the radioactive label was covalently bound exclusively to the beta subunit of the ATPase. Extraction of the modified enzyme from the membrane with chloroform followed by tryptic digestion and separation of peptides by reverse-phase high-pressure liquid chromatography indicated that the radioactive label had been inserted into a peptide fragment that included part of the catalytic site. Covalent modification of catalytic sites by 2-azido-ADP was accompanied by parallel inhibition of both ATP synthesis and ATP hydrolysis by submitochondrial particles. Estimation of the likely amount of F1 participating in the reaction and extrapolation to complete inhibition suggested that modification of no more than a single site was sufficient to block both reactions. The results support suggestions of cooperative interactions between catalytic sites as well as a single catalytic pathway for both enzymic reactions.

Photolabeling of Mitochondrial F1-H+ATPase by 2-Azido[3H]ADP and 8-Azido[3H]ADP Entrapped as Fluorometal Complexes into the Catalytic Sites of the Enzyme

In the presence of ADP and fluorometals, the ATPase activity of the catalytic sector, F1, of beef heart mitochondrial ATPase is strongly inhibited; this inhibition is dependent on the entrapment of ADP-fluoroaluminate complexes into the nucleotide binding sites of F1 [Lunardi, J., Dupuis, A., Garin, J., Issartel, J. P., Michel, L., Chabre, M., & Vignais, P. V. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8958-8962]. We described here the effect of fluoroaluminate on the binding of 2-azido[3H]ADP and 8-azido[3H]ADP to beef heart mitochondrial F1 in the absence and presence of light. When the incubation medium was supplemented with NaF and AlCl3, and maintained in the dark, both 2-azido[3H]ADP and 8-azido[3H]ADP were able to elicit inhibition of F1-ATPase activity, exactly like ADP did. Upon photoirradiation, 2-azido[3H]ADP and 8-azido[3H]ADP bound covalently to F1. Labeling was restricted to the beta subunit of F1, and the same tyrosine residue, beta-Tyr-345, was labeled by either of the photoprobes. This is in contrast with the previous findings that in the absence of fluoroaluminate both the alpha and beta subunits of F1 were photolabeled by 8-azido[3H]ADP, and that two different regions of the beta subunits were labeled, centered on beta-Tyr-345 in the case of 2-azido[3H]ADP [Garin, J., Boulay, F., Issartel, J.P., Lunardi, J., & Vignais, P. V. (1986) Biochemistry 25, 4431-4437] and beta-Tyr-311 that of 8-azido[3H]ADP [Hollemans, M., Runswick, M., Fearnley, I.H., & Walker, J.E. (1983) J. Biol. Chem. 258, 9307-9313].(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Photolabeling of the phosphate binding site of mitochondrial F1-ATPase by [32P]azidonitrophenyl phosphate. Identification of the photolabeled amino acid residues

[32P]Azidonitrophenyl phosphate [( 32P]ANPP) is a photoactivatable analogue of Pi. It competes efficiently with Pi for binding to the F1 sector of beef heart mitochondrial ATPase and photolabels the Pi binding site located in the beta subunit of F1 [Lauquin, G. J. M., Pougeois, R., & Vignais, P. V. (1980) Biochemistry 19, 4620-4626]. By cleavage of the photolabeled beta subunit of F1 with cyanogen bromide, trypsin, and chymotrypsin, bound [32P]ANPP was localized in a fragment spanning Thr 299-Phe 326. By Edman degradation of the radiolabeled tryptic peptide spanning Ile 296-Arg 337, [32P]ANPP was found to be attached covalently by its photoreactive group to Ile 304, Gln 308, and Tyr 311. These results are discussed in terms of a model in which the phosphate group of [32P]ANPP interacts with a glycine-rich sequence of the beta subunit, spanning Gly 156-Lys 162, which is spatially close to the photolabeled Ile 304-Tyr 311 segment of the same subunit.

Rate of chase-promoted hydrolysis of ATP in the high affinity catalytic site of beef heart mitochondrial ATPase.

Incubation of [gamma-32P]ATP with a molar excess of the soluble, homogeneous ATPase from beef heart mitochondria (F1) results in binding of substrate primarily in a single, very high affinity (KA = 10(12) M-1) catalytic site and in a slow rate of hydrolysis characteristic of single site catalysis. Subsequent addition of millimolar concentrations of nonradioactive ATP as a cold chase, sufficient to fill catalytic sites on the enzyme, results in an acceleration of hydrolysis of bound radioactive ATP of as much as 10(6)-fold, that is, to Vmax rates (Cross, R.L., Grubmeyer, C., and Penefsky, H.S. (1982) J. Biol. Chem. 257, 12101-12105). For this reason, it was proposed that the high affinity catalytic site is a normal catalytic site on the molecule. Recently, Bullough et al. (Bullough, D.A., Verburg, J.G., Yoshida, M., and Allison, W.A. (1987) J. Biol. Chem. 262, 11675-11683) reported that when 5 to 20 microM concentrations of nonradioactive ATP were added as a cold chase to an enzyme-substrate complex consisting of F1 and ATP bound to the high affinity catalytic site, hydrolysis of the chase was commensurate with the turnover rate of the enzyme, whereas the hydrolysis of bound ATP was considerably slower. These authors suggested that the high affinity catalytic site on F1 is not a normal catalytic site. This paper shows, in experiments with a rapid mixing-chemical quench apparatus, that hydrolysis of ATP bound in the high affinity catalytic site is accelerated to Vmax rates following addition of 5 microM ATP as a cold chase. Hydrolysis of bound ATP appears to precede that of the chase. The weight of the available evidence continues to support the original suggestion that the high affinity catalytic site of beef heart F1 is a normal catalytic site.

Adenine nucleotide binding sites on beef heart F1 ATPase: photoaffinity labeling of beta-subunit Tyr-368 at a noncatalytic site and beta Tyr-345 at a catalytic site.

2-Azidoadenine [32P]nucleotide was bound specifically at catalytic or noncatalytic nucleotide binding sites on beef heart mitochondrial F1 ATPase. In both cases, photolysis resulted in nearly exclusive labeling of the beta subunit. The modified enzyme was digested with trypsin, and labeled peptides were purified by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis of the major 32P-labeled tryptic fragments showed beta-subunit Tyr-368 to be present at noncatalytic sites and beta Tyr-345 to be present at catalytic sites. From the relationship between the degree of inhibition and extent of modification, it is estimated that one-third of the catalytic sites or two-thirds of the noncatalytic sites must be modified to give near-complete inhibition of catalytic activity.

Dependence of the activity of beef heart mitochondrial adenosinetriphosphatase on the properties of the catalytic metal ion.

Several divalent metal ions were used as kinetic probes of the beef heart mitochondrial adenosinetriphosphatase (F1) under a variety of conditions, and the relationship between the properties of the catalytic metal ion and the catalytic activity of the enzyme was examined. Vmax for ATP hydrolysis was largest when metal ions characterized by intermediate values of acidity of coordinated water molecules (pKa) and metal-nucleotide stability constants (Kstab) were present. As temperature increased, the peak of Vmax vs. pKa (or Kstab) shifted to lower initial values of pKa or Kstab. The solvent deuterium isotope effect on Vmax (DV) was normal and largest when the metal ion present during F1-catalyzed ATP hydrolysis was most acidic and the metal nucleotide stability constant was large. When an active site tyrosine on F1 was nitrated, Vmax was most affected when the metal ion present was least acidic and the metal nucleotide stability constant was small. The isotope effect on V/K (DV/K) was normal, small, and apparently independent of the metal ion present. ADP inhibition of F1-catalyzed ATP hydrolysis is competitive, and the Ki is independent of the metal ion present. The degree of Pi inhibition of F1 is dependent on the metal ion present. The inhibition by Pi is competitive at low temperature and becomes noncompetitive as temperature increases. These and previous results support a mechanism whereby a water molecule coordinated to the metal ion of an enzyme-bound gamma-monodentate metal-ATP complex is deprotonated to begin a series of events whereby a beta,gamma-bidentate metal-ATP complex is produced. Upon hydrolysis, the bond between the metal ion and the beta-phosphate of ADP in the Pi-metal-ADP complex is broken before products (ADP and metal-Pi) are released.

Immunological studies on the beef heart natural ATPase inhibitor: Localization of an antigenic determinant in the inhibitor molecule

Antibodies were raised against the beef heart mitochondrial ATPase inhibitor. This antiserum prevented the ability of the ATPase inhibitor to inhibit the F 1 ATPase activity. Peptide fragments obtained by enzymatic cleavage of the inhibitor protein were tested by immunoblotting or ELISA for their response to the anti-inhibitor antiserum. An antigenic determinant was located in the sequence spanning His 48 to Lys 58 of the inhibitor molecule.

Hydrolysis of adenyl-5-yl imidodiphosphate by beef heart mitochondrial ATPase.

Beef heart mitochondrial ATPase (F1) catalyzes the hydrolysis of the ATP analog adenyl-5-yl imidodiphosphate (AMP-PNP). The reaction products are inorganic phosphate and adenyl-5-yl phosphoramidate (AMP-PN) as determined by HPLC analysis. The hydrolysis occurs in both the presence and absence of added divalent metal ions and is stimulated by potassium. The kinetic properties of the hydrolytic reaction depend markedly on the identity of the added divalent metal. GMP-PNP and AMP-CPP are also hydrolyzed, while AMP-PCP is not. Adenyl-5-yl phosphoramidate is a potent effect of beef heart mitochondrial ATPase activity. Based on these data, a reinterpretation of work based on the assumption that AMP-PNP is not hydrolyzed is presented.

Metal-nucleotide structural characteristics during catalysis by beef heart mitochondrial F1.

This study examined the nature of the metal-nucleotide complexes which serve as substrates, products, and intermediates in the beef heart mitochondrial ATPase reaction. The two methods employed involved the use of phosphorothioate ATP analogs as substrates in the presence of Mg2+ or Cd2+ and the use of substitution inert Cr X ATP complexes (the isolated diastereomers of the bidentate complexes) along with the newly synthesized Cr X ITP complexes as inhibitors of both the F1-ATPase and F1-ITPase activities. Little stereoselectivity was observed in the inhibition of F1-ATPase and F1-ITPase activities by the isolated diastereomers of beta,gamma-bidentate CrATP, while the inhibition by the delta,alpha,beta-bidentate CrADP diastereomer was greater than that of the lambda epimer. gamma-Monodentate CrITP was a weak inhibitor of both the ATPase and ITPase activities, whereas beta,gamma-bidentate CrITP failed to show any inhibition at all up to a concentration of 3.2 mM. When adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) was used as the substrate, (VmSp]/(Vm(Rp] with Mg2+ present was 2.7 at 31 degrees C and 3.5 at 13 degrees C. The (Vm/Km(Sp]/(Vm/Km(Rp] ratios with Mg2+ present were 15.3 at 31 degrees C and 73.3 at 13 degrees C. With Cd2+ present, the (Vm(Sp]/(Vm(Rp] ratios were 0.81 and 0.65 at 31 and 13 degrees C, respectively. The (Vm/Km(Sp]/(Vm/Km(Rp] ratios with Cd2+ present were 1.17 at 31 degrees C and 1.34 at 13 degrees C. The large activation energy observed for the isomers of CdATP beta S was not observed for MgATP beta S, MgATP, or CdATP. The Vm for Cd adenosine 5'-O-thiotriphosphate (ATP gamma S) hydrolysis was the largest of all the metal-phosphorothioate nucleotide complexes, while that for MgATP gamma S was the smallest. The results are interpreted in terms of a catalytic model for F1-catalyzed nucleotide hydrolysis describing metal-nucleotide chelation during the reaction.

The effect of Co(III)(NH 3) 4ATP on the kinetics of beef heart mitochondrial ATPase

Bidentate cobalt(III)tetraamine adenosine triphosphate [Co(NH 3) 4ATP] was investigated as an inhibitor of the beef heart mitochondrial F 1-ATPase. The compound was found to have a mixed noncompetitive mechanism with a K i of 0.4 m m and an α of 1.4 during ATP hydrolysis. Co(NH 3) 4ATP also noncompetitively inhibited ATP hydrolysis in the presence of bicarbonate. ITP hydrolysis was similarly affected. Co(NH 3) 4ATP was also used in dual inhibitor studies with adenylylimidodiphosphate (AMP-PNP) and azide; it was found to be mutually exclusive with AMP-PNP and azide. The compound also protected the F 1 from modification by 4-chloro-7-nitroben-zofurazan. These results are discussed in terms of the regulation of the ATP hydrolysis reaction.

Metal interactions with beef heart mitochondrial ATPase.

Atomic absorption and electron paramagnetic resonance spectroscopy were used to study the metal binding sites of beef heart mitochondrial ATPase (F1). Quantitative and qualitative properties of these sites are described. Two different separation techniques were able to distinguish two very tight sites from one tight (easily exchangeable) metal binding site on F1. Of these sites, two are specific for magnesium while one can be substituted with Mn2+, Co2+, or Zn2+. When MgAMP-PNP was incubated with F1, a fourth metal was bound to the enzyme. The carboxyl group modified by dicyclohexylcarbodiimide is shown not to be involved in binding of any of the tightly bound metals. Qualitative properties of the metal binding sites using the Mn2+-enzyme complex as a probe were ascertained using EPR at pH 6.8 and 8.0. CrATP and Mn2+ appear to bind to different metal sites on F1. The possible role of the metals in regulation of catalysis, and their relation to nucleotide binding is discussed.

Interaction of azide with beef heart mitochondrial ATPase

This study examined the inhibition of azide as a probe of the magnesium regulation of beef heart mitochondrial ATPase (F 1) catalysis. Azide elicited a slow hysteretic effect on both ATP and ITP hydrolysis of F 1. This hysteretic effect was shown to be due to the consecutive binding of magnesium and azide, and to be independent of catalytic turnover. The azide binding site was also shown to be separate from the anion binding HCO 3 − site on F 1. The results presented indicate that metal binding is important in the inhibition of the hydrolytic activity and regulation of F 1. A model is presented which is consistent with the hysteretic inhibition of F 1 by azide, in which there is a slow equilibration between free enzyme and the enzyme-magnesium-azide complex.

The oligomycin sensitivity conferring protein (OSCP) of beef heart mitochondria: studies of its binding to F1 and its function.

The binding of "oligomycin sensitivity conferring protein" (OSCP) to soluble beef-heart mitochondrial ATPase (F1) has been investigated. OSCP forms a stable complex with F1, and the F1 X OSCP complex is capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F1- and OSCP-depleted submitochondrial particles. The F1 X OSCP complex retains 50% of its ATPase activity upon cold exposure while free F1 is inactivated by 90% or more. Both free F1 and the F1 X OSCP complex release upon cold exposure a part--probably 1 out of 3--of their beta subunits; whether alpha subunits are also lost is uncertain. The cold-treated F1 X OSCP complex is still capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F1- and OSCP-depleted particles. OSCP also protects F1 against modification of its alpha subunit by mild trypsin treatment. This finding together with the earlier demonstration that trypsin-modified F1 cannot bind OSCP indicates that OSCP binds to the alpha subunit of F1 and that F1 contains three binding sites for OSCP. The results are discussed in relation to the possible role of OSCP in the interaction of F1 with the membrane sector of the mitochondrial ATPase system.

Pre-steady-state kinetics of beef heart mitochondrial ATPase

The pre-steady-state kinetics of beef heart mitochondrial ATPase (F 1) were examined. F 1 was found to exhibit hysteretic behavior when hydrolyzing ATP. The hysteretic property was expressed as an activation process which occurred when the enzyme was mixed with its substrate, MgATP. Many catalytic turnovers were required before the activation was complete. The lag in hydrolysis increased hyperbolically as the concentration of enzyme increased. Passage of F 1 through Sephadex G25 eliminated the activation process. Several kinetically distinct possibilities for explaining these data, including multiple nucleotide dissociations, enzyme conformational changes, and regulatory site interactions, are discussed. The enzyme was apparently able to recognize nucleotide in a noncatalytic manner, as evidenced by the fact that F 1 preincubated with ADP in the absence of substrate achieved partial activation (smaller lag times) before being introduced to substrate. ADP is also a time-dependent inhibitor, exhibiting a slow hysteretic inhibition in addition to immediate competitive inhibition.

Hydrophobic and ionic effects upon the electrophoretic mobilities of the subunits of coupling factor 1 from mitochondria

A sodium dodecyl sulfate (SDS)-urea polyacrylamide gel system was used to investigate certain properties of the subunits of the beef heart mitochondrial ATPase, (native F1, nF1). By examining the affects of urea concentration and acrylamide concentration upon the electrophoretic mobilities of the polypeptides comprising the nF1 enzyme, we have obtained conditions under which all five subunits are simultaneously resolved when the discontinuous buffer system of Laemmli is used ( U. K. Laemmli (1970) Nature (London) 277, 680–685). The determination of the apparent molecular weights by analysis of Ferguson plots ( K. A. Ferguson (1964) Metabolism 13, 985–1002) revealed that the addition of urea to the SDS gels resulted in a decrease in the apparent molecular weight of the β subunit. A dramatic increase in the apparent molecular weight of the δ subunit was also brought about by the presence of urea in the SDS gels. In addition, the apparent molecular weight of both the α and the β subunits was dependent upon the acrylamide concentration used, indicating that these subunits contain either areas highly resistant to denaturation by the combined action of urea and SDS, or covalent modifications leading to anomalous electrophoretic mobility. The results of experiments in which urea analogs were used indicate that the interactions of urea with the β subunit involve the formation of hydrogen bonds between urea and regions of this subunit. On the other hand, the interactions of urea with the δ subunit are primarily of a hydrophobic nature, suggesting that these interactions could involve domains of the δ subunit required for binding of the coupling factor to the mitochondrial membrane.

Mechanism of ATP hydrolysis by beef heart mitochondrial ATPase. Rate constants for elementary steps in catalysis at a single site.

Mechanism of ATP hydrolysis by beef heart mitochondrial ATPase. Rate constants for elementary steps in catalysis at a single site.

Mechanism of ATP hydrolysis by beef heart mitochondrial ATPase. Rate enhancements resulting from cooperative interactions between multiple catalytic sites.

Mechanism of ATP hydrolysis by beef heart mitochondrial ATPase. Rate enhancements resulting from cooperative interactions between multiple catalytic sites.

Catalytic properties of beef heart mitochondrial ATPase modified with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Evidence for catalytic site cooperativity during ATP synthesis.

Catalytic properties of beef heart mitochondrial ATPase modified with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Evidence for catalytic site cooperativity during ATP synthesis.