On the location and function of the noncatalytic sites on the bovine heart mitochondrial F1-ATPase

Modification of Tyr-345 at a catalytic site in a single beta subunit of the bovine heart mitochondrial F1-ATPase (MF1) by 5'-p-fluorosulfonylbenzoylinosine did not affect subsequent labeling of noncatalytic sites at Tyr-368 and His-427 in three copies of the beta subunit by 5'-p-fluorosulfonylbenzoyladenosine (FSBA). These results clearly show that the beta subunit contains at least parts of the catalytic and noncatalytic nucleotide binding sites. Inactivation of MF1 by 96% with FSBA was accompanied by a decrease in the endogenous ADP content from 1.86 to 0.10 mol per mol of MF1. Decrease in the endogenous ADP content during the inactivation of the enzyme with FSBA paralleled loss in activity in a manner which suggests that the reaction of FSBA with an open noncatalytic site promoted release of ADP from another noncatalytic site until the third site reacted with FSBA. Two pKa values of about 5.9 and 7.6 were observed on the acid side of the pH optimum in the pH-rate profile for ATP hydrolysis catalyzed by MF1 in neutral acid buffers. In contrast, a single pKa of 5.9 was present in the pH-rate profile for ITP hydrolysis catalyzed by the enzyme in the same buffers. The augmented rate observed for ATP hydrolysis at pH 8.0, over that observed at pH 6.5, was lost as the enzyme was inactivated by FSBA in a manner suggesting that modulation is lost as the third noncatalytic site is modified. This suggests that ATP hydrolysis by MF1 is modulated in a pH-dependent manner by ATP binding to an open noncatalytic site. Two other modulations associated with binding of adenine nucleotides to noncatalytic sites, ADP-induced hysteretic inhibition and apparent negative cooperativity reflected by the Hill coefficient for the hydrolysis of 50-3000 microM ATP at pH 8.0, also disappeared as the third noncatalytic site reacted with FSBA.     

Inactivation of the bovine heart mitochondrial F1-ATPase by 5'-p-fluorosulfonylbenzoyl[3H]inosine is accompanied by modification of tyrosine 345 in a single beta subunit

The inactivation of the bovine heart mitochondrial F1-ATPase by 5'-p-fluorosulfonylbenzoylinosine (FSBI) proceeds with pseudo-first order kinetics. The rate of inactivation increased from pH 7 to 9 revealing a pKa of about 8.2. When a tryptic digest of the enzyme which had been inactivated with 5'-p-fluorosulfonylbenzoyl[3H]inosine ([3H]FSBI) was submitted to reversed phase high pressure liquid chromatography, a single major peak of radioactivity, T1, was resolved. Amino acid sequence analysis of purified peptide fragments derived from T1 showed that the modification of beta-Tyr-345 is responsible for inactivation of the enzyme. Complete inactivation of the enzyme by [3H]FSBI is estimated to proceed with modification of 0.8 mol of beta-Tyr-345/mol of enzyme. Another notable observation is that inosine triphosphatase (ITPase) activity catalyzed by F1 from bovine heart mitochondria is much more sensitive to inactivation by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) than is ATPase activity. Whereas complete inactivation of ATPase activity by FSBA has been shown to proceed with the mutually exclusive modification of Tyr-368 or His-427 in all three copies of the beta subunit (Bullough, D. A., and Allison, W. S. (1986) J. Biol. Chem. 261, 5722-5730), it is shown here that complete inactivation of ITPase activity by FSBA is accompanied by modification of these residues in only one copy of the beta subunit. Inactivation of both the ATPase and ITPase activities of the enzyme by FSBI proceeds with modification of Tyr-345 in a single copy of the beta subunit.     

Separate beta subunits are derivatized with 14C and 3H when the bovine heart mitochondrial F1-ATPase is doubly labeled with 7-chloro-4-nitro[14C]benzofurazan and 5'-p-fluorosulfonylbenzoyl[3H]inosine.

Tyrosine residues 311 and 345 of the beta subunit of the bovine heart mitochondrial F1-ATPase (MF1) are present on the same peptide when the enzyme is fragmented with cyanogen bromide. Maximal inactivation of MF1 with 7-chloro-4-nitro[14C]benzofurazan [( 14C]Nbf-Cl) derivatizes tyrosine-311 in a single beta subunit. Cyanogen bromide digests of MF1 containing the [14C]Nbf-O-derivative of tyrosine-beta 311 were submitted to reversed-phase HPLC, with and without prior reduction of the nitro group on the incorporated reagent with dithionite. The retention time of the radioactive cyanogen bromide peptide was shifted substantially by reduction. When a cyanogen bromide digest of MF1 inactivated with 5'-p-fluorosulfonylbenzoyl[3H]inosine [( 3H]FSBI), which proceeds with derivatization of tyrosine-345 in a single beta subunit, was submitted to HPLC under the same conditions, the fragment labeled with 3H eluted with the same retention time as the [14C]Nbf-O-derivative before reduction. Doubly labeled enzyme was prepared by first derivatizing Tyr-beta 311 with [14C]Nbf-Cl and then derivatizing tyrosine-beta 345 with [3H]FSBI with and without reducing the [14C]Nbf-O-derivative of tyrosine-beta 311 with dithionite before modification with [3H]FSBI. The doubly labeled enzyme preparations were digested with cyanogen bromide and submitted to HPLC. The 14C and 3H in the cyanogen bromide digest prepared from doubly labeled enzyme not submitted to reduction eluted together. In contrast, the 14C and 3H in the digest prepared from doubly labeled enzyme which had been reduced eluted separately. From these results it is concluded that different beta subunits are derivatized when MF1 is doubly labeled with [14C]Nbf-Cl and [3H]FSBI.     

The varied responses of different F1-ATPases to chlorpromazine

The effects of chlorpromazine on various properties of the F1-ATPases from bovine heart mitochondria (MF1), the plasma membranes of Escherichia coli (EF1), and plasma membranes of the thermophilic bacterium PS3 (TF1) have been examined. While chlorpromazine inhibited MF1 with an I0.5 of about 50 microM and EF1 with an I0.5 of about 150 microM at 23 degrees C, the ATPase activity of TF1 was stimulated by chlorpromazine concentrations up to 0.6 mM at this temperature. Maximal activation of about 20% was observed at 0.2 mM chlorpromazine at 23 degrees C. Chlorpromazine concentrations greater than 0.6 mM inhibited TF1 at 23 degrees C. At 37 degrees C the ATPase activity of TF1 was doubled in the presence of 0.5 mM chlorpromazine, the concentration at which maximal stimulation was observed at this temperature. Chlorpromazine inhibited the rate of inactivation of EF1 by dicyclohexylcarbodiimide (DCCD) at 23 degrees C and pH 6.5. Concentrations of chlorpromazine which inhibited the ATPase activity of TF1 at pH 7.0 accelerated the rate of inactivation of the enzyme by DCCD at pH 6.5, while lower concentrations of the phenothiazine, which stimulated the ATPase, had no effect on DCCD inactivation. Chlorpromazine concentrations up to 1.0 mM had no effect on the rate of inactivation of TF1 by DCCD at 37 degrees C and pH 6.5. Chlorpromazine at 0.5 mM accelerated the rate of inactivation of MF1 by 5'-p-fluorosulfonylbenzoyladenosine (FSBA), while it slowed the rate of inactivation of EF1 by FSBA. The inactivation of TF1 by FSBA in the absence of chlorpromazine was complex and was not included in this comparison. Chlorpromazine protected MF1 and EF1 against cold inactivation. Whereas 100 microM chlorpromazine afforded about 90% stabilization of MF1 at 4 degrees C, only about 30% stabilization of EF1 was observed under the same conditions in the presence of 400 microM chlorpromazine. Each of the ATPases was inactivated by the structural analog of chlorpromazine, quinacrine mustard. Whereas 5 mM ATP and 5 mM ADP protected MF1 and TF1 against inactivation by 0.5 mM quinacrine mustard, the rate of inactivation of EF1 by quinacrine mustard was accelerated fourfold by 5 mM ATP and slightly accelerated by 5 mM ADP.     

5′-p-Fluorosulfonylbenzoyladenosine as an ATP site affinity probe for Na+,K+-ATPase

We have investigated the suitability of 5′-p-fluorosulfonylbenzoyladenosine (FSBA) as an ATP site affinity probe for the canine kidney Na⁺,K⁺-ATPase. The purified enzyme is slowly inactivated by this compound in suitable buffers, losing about half of its activity over a two-hour period. The rate of inactivation is more rapid in 0.1 M KCl than in 0.1 M NaCl. Low concentrations of ATP protect the enzyme against inactivation, with half-maximal effects at 4 μM ATP in 0.1 M NaCl and 350 μM ATP in 0.1 M KCl. ADP also protects against FSBA inhibition, but AMP is ineffective when present at 100 μM levels. This pattern is consistent with the previously described nucleotide specificity of the Na⁺,K⁺-ATPase. Addition of protective amounts of ATP after inactivation has occurred does not restore enzyme activity, indicating that inhibition is irreversible. Measurement of the concentration-dependence of FSBA inactivation suggests an apparent K_d for binding of this compound well above 1 mM, the solubility limit of the analog. This finding is reinforced by the failure of 1 mM FSBA to compete effectively with ATP for the high-affinity ATP site of the enzyme. Nevertheless, attachment of the analog to this site is indicated by its ability to prevent [³H]-ADP binding in proportion to the number of sites it has inactivated. Studies with [³H]-FSBA show that about 1 mole of the analog attaches specifically to the α subunit per mole of enzyme inactivated. A similar amount of nonspecific labeling also occurs with negligible effect on enzyme activity. These findings suggest that FSBA may be useful in probing the topography of the high-affinity ATP binding site of the Na⁺,K⁺-ATPase and related enzymes.     

Sequence of the radioactive tryptic peptide obtained after inactivating the F1-ATPase of the thermophilic bacterium PS3 with 5'-p-fluorosulfonylbenzoyl[3H]adenosine at 65 degrees C

Following a lag of about 30 min, the F1-ATPase from the thermophilic bacterium, PS3 (TF1), was inactivated slowly by 0.8 mM 5'-p-fluorosulfonylbenzoyladenosine (FSBA) at 23 degrees C and pH 7.0. When the enzyme was treated with 0.2 mM FSBA at pH 7.0 and 23 degrees C for 15 min and gel-filtered, no enzyme activity was lost. However, the lag in inactivation was abolished when the enzyme was subsequently incubated with 2.0 mM FSBA at 23 degrees C in the pH range from 6.8 to 10.0. The pH-inactivation profile obtained under these conditions revealed a pK alpha of about 9.3 which was associated with the inactivation. When pretreated TF1 was inactivated at 23 degrees C with [3H]FSBA by about 90%, greater than 20 mol of [3H]SBA was incorporated per mole of enzyme. TF1 was inactivated rapidly by 0.8 mM FSBA at pH 6.4 and 65 degrees C, and no lag was observed. Following inactivation of TF1 with 0.8 mM [3H]FSBA at 65 degrees C and pH 6.4, about 10 mol of [3H]SBA was incorporated per mole of enzyme. When a tryptic digest of the labeled enzyme was fractionated by reversed-phase high-performance liquid chromatography, a single major radioactive peptide was isolated. When subjected to automatic Edman degradation, this peptide was shown to have the amino acid sequence: A-L-A-P-E-I-V-G-E-E-H-X-Q-V-A-R, where X indicates that a phenylthiohydantoin derivative was not detected in cycle 12. However, from the DNA sequence of the gene encoding the subunit of TF1 (Y. Kagawa, M. Ishizuka, T. Saishu, and S. Nakao (1985) Abstracts International Symposium on Energy Transducing ATPases, Kobe, Japan, p. 84), this position has been shown to be occupied by tyrosine. This tyrosine is homologous with beta-Tyr-368 of the bovine mitochondrial F1-ATPase (MF1) the modification of which is responsible for the inactivation MF1 by FSBA.     

Adenine nucleotide binding at a noncatalytic site of mitochondrial F1-ATPase accelerates a Mg(2+)- and ADP-dependent inactivation during ATP hydrolysis.

The evidence is presented that the ADP- and Mg(2+)-dependent inactivation of MF1-ATPase during MgATP hydrolysis requires binding of ATP at two binding sites: one is catalytic and the second is noncatalytic. Binding of the noncatalytic ATP increases the rate of the inactive complex formation in the course of ATP hydrolysis. The rate of the enzyme inactivation during ATP hydrolysis depends on the medium Mg2+ concentration. High Mg2+ inhibits the steady-state activity of MF1-ATPase by increasing the rate of formation of inactive enzyme-ADP-Mg2+ complex, thereby shifting the equilibrium between active and inactive enzyme forms. The Mg2+ needed for MF1-ATPase inactivation binds from the medium independent from the MgATP binding at either catalytic or noncatalytic sites. The inhibitory ADP molecule arises at the MF1-ATPase catalytic site as a result of MgATP hydrolysis. Exposure of the native MF1-ATPase with bound ADP at a catalytic site to 1 mM Mg2+ prior to assay inactivates the enzymes with kinact 24 min-1. The maximal inactivation rate during ATP hydrolysis at saturating MgATP and Mg2+ does not exceed 10 min-1. The results show that the rate-limiting step of the MF1-ATPase inactivation during ATP hydrolysis with excess Mg2+ precedes binding of Mg2+ and likely is the rate of formation of enzyme with ADP bound at the catalytic site without bound P(i). This complex binds Mg2+ resulting in inactive MF1-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the ATP binding sites of the carbamyl phosphate synthetase domain of the Syrian hamster multifunctional protein CAD by affinity labeling with 5'-[p-(fluorosulfonyl)benzoyl]adenosine.

The ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA) was used to chemically modify the ATP binding sites of the carbamyl phosphate synthetase domain of CAD, the multifunctional protein that catalyzes the first steps in mammalian pyrimidine biosynthesis. Reaction of CAD with FSBA resulted in the inactivation of the ammonia- and glutamine-dependent CPSase activities but had no effect on its glutaminase, aspartate transcarbamylase, or dihydroorotase activities. ATP protected CAD against inactivation by FSBA whereas the presence of the allosteric effectors UTP and PRPP afforded little protection, which suggests that the ATP binding sites were specifically labeled. The inactivation exhibited saturation behavior with respect to FSBA with a K1 of 0.93 mM. Of the two ATP-dependent partial activities of carbamyl phosphate synthetase, bicarbonate-dependent ATPase was inactivated more rapidly than the carbamyl phosphate dependent ATP synthetase, which indicates that these partial reactions occur at distinct ATP binding sites. The stoichiometry of [14C]FSBA labeling showed that only 0.4-0.5 mol of FSBA/mol of protein was required for complete inactivation. Incorporation of radiolabeled FSBA into CAD and subsequent proteolysis, gel electrophoresis, and fluorography demonstrated that only the carbamyl phosphate synthetase domain of CAD is labeled. Amino acid sequencing of the principal peaks resulting from tryptic digests of FSBA-modified CAD located the sites of FSBA modification in regions that exhibit high homology to ATP binding sites of other known proteins. Thus CAD has two ATP binding sites, one in each of the two highly homologous halves of the carbamyl phosphate domain which catalyze distinct ATP-dependent partial reactions in carbamyl phosphate synthesis.     

On the subunit stoichiometry of the F1-ATPase and the sites in it that react specifically with p-fluorosulfonylbenzoyl-5'-adenosine.

During the inactivation of the nucleotide-free F1-ATPase at pH 7.0, by p-fluorosulfonyl[14C]benzoyl-5'-adenosine ([14C]FSBA) in the presence of 20% glycerol, about 4.5 g atoms of 14C are incorporated/350,000 g of enzyme. Isolation of the subunits has shown: (a) over 90% of the incorporated label is associated with the alpha and beta subunits; (b) the amount of label incorporated into the alpha subunit is about 0.5 g atoms/mol which is nonspecifically associated with a number of tyrosine and lysine residues; (c) the amount of radioactivity incorporated into the beta subunit is about 0.9 g atoms/mol which correlates with the degree of inactivation of the enzyme and resides on a single tyrosine residue; (d) up to 2.2 mol of alpha subunit have been isolated from each mole of inactivated enzyme; and (e) about 2 mol of beta subunit have been isolated from each mole of inactivated enzyme. These results account for the incorporation of 4.5 g atoms of 14C which are incorporated/mol of ATPase during inactivation if there are three copies each of the alpha and beta subunit present in the enzyme. It has also been shown that 4-chloro-7-nitrobenzofurazan (NBD-Cl) and FSBA react with different tyrosine residues when they inactivate the ATPase. In addition, it has been shown that the ATPase inactivated with FSBA retains the capacity to bind up to 2.2 mol of [14C]ADP/350,000 g of enzyme.     

The ATP-binding site of brain phosphatidylinositol 4-kinase PI4K230 as revealed by 5'-p-fluorosulfonylbenzoyladenosine

The ATP-binding site of purified bovine brain phosphatidylinositol 4-kinase 230 (PI4K230) was studied by its reaction with 5'-p-fluorosulfonylbenzoyladenosine (FSBA), an ATP-like alkylating reagent. Four hundred to eight hundred micromolar FSBA inactivated PI4K230 specifically with apparently first-order kinetics and resulted in 50% loss of enzyme activity in 36--130 min. The specificity of the reaction with FSBA was demonstrated by the lack of inactivation with 5'-p-fluorosulfonylbenzoyl chloride and by protection with ATP and ATP analogues against inactivation. Most ATP analogues competed with FSBA inactivation in order of their increasing hydrophobicity, parallel to their inhibitory potency in activity measurements. The specific binding of FSBA to PI4K230 was demonstrated also by Western-blot experiments. These results suggest that FSBA-reactive group(s) involved in the enzyme activity are located near to the ATP-binding site in a hydrophobic region of native PI4K230. Experiments with site-directed mutagenesis indicate that the conserved Lys-1792 plays essential role in the enzyme activity and serves as one target of affinity labelling by FSBA. Prevention of both Lys-1792-directed and Lys-1792-independent binding of FSBA by Cibacron Blue 3GA suggest that these sites are located spatially close to each other.     

The ionic track in the F1-ATPase from the thermophilic Bacillus PS3

Only beta-beta cross-links form when the alpha(3)(betaE(395)C)(3)gammaK(36)C (MF(1) residue numbers) double mutant subcomplex of TF(1), the F(1)-ATPase from the thermophilic Bacillus PS3, is slowly inactivated with CuCl(2) in the presence or absence of MgATP. The same slow rate of inactivation and extent of beta-beta cross-linking occur upon treatment of the alpha(3)(betaE(395)C)(3)gamma single mutant subcomplex with CuCl(2) under the same conditions. In contrast, the alpha(3)(betaE(395)C)(3)gammaR(33)C and alpha(3)(betaE(395)C)(3)gammaR(75)C double mutant subcomplexes of TF(1) are rapidly inactivated by CuCl(2) under the same conditions that is accompanied by complete beta-gamma cross-linking. The ATPase activity of each mutant enzyme containing the betaE(395)C substitution is stimulated to a much greater extent by the nonionic detergent lauryldimethylamine oxide (LDAO) than wild-type enzyme, whereas the ATPase activities of the gammaR(33)C, gammaK(36)C, and gammaR(75)C single mutants are stimulated to about the same extent as wild-type enzyme by LDAO. This indicates that the E(395)C substitution in the (394)DELSEED(400) segment of beta subunits increases propensity of the enzyme to entrap inhibitory MgADP in a catalytic site during turnover. These results are discussed in perspective with (i) the ionic track predicted from molecular dynamics simulations to operate during energy-driven ATP synthesis by MF(1), the F(1)-ATPase from bovine heart mitochondria [Ma, J., Flynn, T. C., Cui, Q., Leslie, A. G. W., Walker, J. E., and Karplus, M. (2002) Structure 10, 921-931]; and (ii) the possibility that the betaE(395)C substitution might induce a global effect that alters affinity of noncatalytic sites for nucleotides or alters communication between noncatalytic sites and catalytic sites during ATP hydrolysis.     

Chemical modification of Salmonella typhimurium phosphoribosylpyrophosphate synthetase with 5'-(p-fluorosulfonylbenzoyl)adenosine. Identification of an active site histidine

Liquid chromatographic procedures have been developed for rapidly locating the site of reaction of chemical modification reagents with Salmonella typhimurium 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase. The enzyme was reacted with the active site-directed reagent 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA). FSBA bound to the enzyme with an apparent KD of 1.7 +/- 0.4 mM. The enzyme was inactivated during the reaction, and a limiting stoichiometry of 1.2 mol of FSBA/mol of enzyme subunit corresponded to complete inactivation. Inclusion of ATP in the reaction protected the enzyme from inactivation and incorporation of the reagent. Inclusion of ribose 5-phosphate increased the rate of reaction of PRPP synthetase with FSBA. Amino acid analyses of acid hydrolysates of modified enzyme failed to detect any known FSBA-amino acid adducts. Tryptic digestion of 5'-(p-fluorosulfonylbenzoyl)-[3H]adenosine-modified enzyme at pH 7.0 yielded a single radioactive peptide. The peptide, TR-1, was subjected to combined V8 and Asp-N protease digestion, and a single radioactive peptide was isolated. This radioactive peptide yielded the sequence Asp-Leu-His-Ala-Glu, which corresponded to amino acid residues 128-132 in S. typhimurium PRPP synthetase. No radioactivity was associated with any of the phenylthiohydantoin-amino acid fractions, all of which were recovered in good yield. A majority of the radioactivity was found in the waste effluent (64%) and on the glass fiber filter loaded into the sequenator (23%). The lability of the modification and the sequence of this peptide indicate His130 as the site of reaction with FSBA.     

Three copies of the beta subunit must be modified to achieve complete inactivation of the bovine mitochondrial F1-ATPase by 5'-p-fluorosulfonylbenzoyladenosine

The modification of both beta-Tyr-368 and beta-His-427 can be correlated with the loss of activity observed when the bovine mitochondrial F1-ATPase is inactivated with 5'-p-fluorosulfonylbenzoyl[3H]adenosine ([3H]FSBA). At pH 8.0, where the rate of inactivation is fast, beta-Tyr-368 is modified predominantly, while at pH 6.0, where the rate of inactivation is slow, beta-His-427 is modified predominantly. At pH 7.0, the 2 residues are modified with about equal efficiency. When the F1-ATPase was inactivated by 80% at pH 6.5, 7.0, and 7.5, the sum of radioactivity incorporated into beta-Tyr-368 and beta-His-427 was 1.99, 1.87, and 1.82 mol of label incorporated per mol of enzyme, respectively. Examination of the rate of inactivation of the enzyme by FSBA as a function of pH revealed two pKa values, one of about 7.6 associated with the modification of beta-Tyr-368 and the other of about 5.8 associated with the modification of beta-His-427. The inactivation of the F1-ATPase by FSBA exhibited an initial fast rate followed by a slower rate in triethanolamine-HCl, pH 7.0. In contrast, only a single rate, equivalent to the fast phase of inactivation in the absence of phosphate, was observed in 0.2 M phosphate, pH 7.0. The dependence of this stimulation on phosphate concentration is sigmoidal with half-maximal stimulation occurring at approximately 160 mM. The ratio of 3H incorporated into beta-Tyr-368 to that incorporated into beta-His-427 was approximately the same during the fast and slow phases of inactivation in triethanolamine-HCl, pH 7.0. Approximately the same ratio was observed when the enzyme was modified during the single phase of inactivation exhibited in the presence of 0.2 M phosphate, pH 7.0. The sum of the 3H incorporated into beta-Tyr-368 and beta-His-427 during inactivation of the F1-ATPase from bovine heart mitochondria by [3H]FSBA in the presence and absence of phosphate was linear and extrapolated to a value of about 2.6 residues modified on complete inactivation of the enzyme. From these data, it is concluded that FSBA binds to a single binding site on the beta subunits of the enzyme where it reacts with either beta-Tyr-368 or beta-His-427 in mutually exclusive reactions. All three beta subunits must be modified in this manner for complete inactivation to be observed.     

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)     

Identification of the reactive cysteinyl residue and ATP binding site in Bacillus cereus glutamine synthetase by chemical modification

Bacillus cereus glutamine synthetase was modified by reaction with a fluorescent SH reagent, N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS), or an ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The locations of the specific binding sites of these reagents were identified. IAEDANS inactivated Mg2(+)-dependent activity and activated Mn2(+)-dependent activity. FSBA inactivated only Mn2(+)-dependent activity. Mg2+ plus Mn2(+)-dependent activity was inactivated by IAEDANS or FSBA. Amino acid sequence analysis of the single AEDANS-labeled proteolytic fragment showed the cysteinyl residue at position 306 to be the site of modification. Cys 306 is one of three cysteines that are unique to Bacillus glutamine synthetase. The result suggested that the cysteine has a role in the active site of the enzyme. We also report that the amino acid residue modified by FSBA was the lysyl residue at position 43.     

Mitochondrial nicotinamide nucleotide transhydrogenase: active site modification by 5'-[p-(fluorosulfonyl)benzoyl]adenosine

Membrane-bound and purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) was inhibited by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), which is an analogue of TH substrates and their competitive inhibitors, namely, 5'-, 2'-, or 3'-AMP. NAD(H) and analogues, NADP, 5'-AMP, 5'-ADP, and 2'-AMP/3'-AMP mixed isomers protected TH against inhibition by FSBA, but NADPH accelerated the inhibition rate. In the absence of protective ligands or in the presence of NADP, FSBA appeared to modify the NAD(H) binding site of TH, because, unlike unmodified TH, the enzyme modified by FSBA under these conditions did not bind to an NAD-affinity column (NAD-agarose). However, when the NAD(H) binding site of TH was protected in the presence of 5'-AMP or NAD, then FSBA modification resulted in an inhibited enzyme that did bind to NAD-agarose, suggesting FSBA modification of the NADP(H) binding site or an essential residue outside the active site. [3H]FSBA was covalently bound to TH, and complete inhibition corresponded to the binding of about 0.5 mol of [3H]FSBA/mol of TH. Since purified TH is known to be dimeric in the isolated state, this binding stoichiometry suggests half-of-the-sites reactivity. A similar binding stoichiometry was found earlier for complete inhibition of TH by [14C]DCCD [Phelps, D.C., & Hatefi, Y. (1984) Biochemistry 23, 4475-4480]. The active site directed labeling of TH by radioactive FSBA should allow isolation of appropriate peptides for sequence analysis of the NAD(H) and possibly the NADP(H) binding domains.     

Stoichiometry of labeling of myosin's proteolytic fragments by a purine disulfide analog of adenosine triphosphate.

A site-specific analog of ATP, 6,6'-dithiobis (inosinyl imidodiphosphate (S2P-PNP), inactivates the ATPase activities of myosin's proteolytic fragments, heavy meromyosin (HMM) and subfragment one (SF1), by formation of mixed disulfides between the 6 position of the purine ring and certain key cysteines. The stoichiometry of the reaction was determined by quantitatively displacing the thiopurine nucleotides from the labeled enzymes with sodium[14-C]cyanide. The thiocyanatoenzyme formed regained 25 percent of the original activity showing that the cysteines modified were not essential for catalysis. The rate of uptake of label paralleled the rate of inactivation. HMM was completely inactivated when 4 mol of thiopurine nucleotide was bound. SF1 made by a papain digestion of myosin incorporarted 2 mol of thiopurine nucleotide when completely inactivated. Having adenylyl imidodiphosphate, areversible competitive inhibitor of myosin's ATPase, present during the inactivation of HMM by S2P-PNP demonstrated that only one cysteine per head needed to be blocked to inactivate the enzyme. Moreover, SF1 made by a trypsin digest of HMM was completely inactivated when only 1.1 mol of the thiopurine nucleotide bound again indicating that blocking only a single cysteine per head was sufficient to cause inactivation. This sulfhydryl is thought to be at an ATP binding site distinct from the ATPase site. The properties of this second ATP binding site are consistent with it being an ATP regulatory site.     

Affinity labeling of phosphoglycerate kinase by 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine

Yeast phosphoglycerate kinase is irreversibly inactivated upon incubation with 5'-[p-(fluorosulfonyl)-benzoyl]-1-N6-ethenoadenosine (5'-FSB epsilon A), an analogue to the nucleotide substrate. Marked protection against inactivation occurs with MgATP, ATP, MgADP, ADP, and 3-phosphoglycerate, suggesting that a part of the catalytic center is modified. The time dependence of the inactivation is characterized by a nonlinear kinetic profile. Curve fitting of various models for ligand binding to the enzyme suggested a two-site model. Modification of one of the sites appears to protect the catalytically essential site from modification. Stoichiometric studies show that the relationship between moles of 5'-FSB epsilon A incorporated per mole of enzyme and the residual enzymatic activity also shows nonlinear behavior. An extrapolated value of 1.5 mol of bound label/mol of enzyme corresponds to complete inactivation. The apparent overall pseudo first-order rate constant for the reaction between phosphoglycerate kinase and 5'-FSB epsilon A, as well as the separate rate constants for the modification, exhibit saturation behavior with respect to the concentration of 5'-FSB epsilon A, indicative of a rapid reversible binding of the reagent to the enzyme prior to modification.     

The characteristics and effect on catalysis of nucleotide binding to noncatalytic sites of chloroplast F1-ATPase.

The recent finding that the presence of ATP at non-catalytic sites of chloroplast F1-ATPase (CF1) is necessary for ATPase activity (Milgrom, Y. M., Ehler, L. L., and Boyer, P. D. (1990) J. Biol. Chem. 265,18725-18728) prompted more detailed studies of the effect of noncatalytic site nucleotides on catalysis. CF1 containing at noncatalytic sites less than one ADP or about two ATP was prepared by heat activation in the absence of Mg2+ and in the presence of ADP or ATP, respectively. After removal of medium nucleotides, the CF1 preparations were used for measurement of the time course of nucleotide binding from 10 to 100 microM concentrations of 3H-labeled ADP, ATP, or GTP. The presence of Mg2+ strongly promotes the tight binding of ADP and ATP at noncatalytic sites. For example, the ADP-heat-activated enzyme in presence of 1 mM Mg2+ binds ADP with a rate constant of 0.5 x 10(6) M-1 min-1 to give an enzyme with two ADP at noncatalytic sites with a Kd of about 0.1 microM. Upon exposure to Mg2+ and ATP the vacant noncatalytic site binds an ATP rapidly and, as an ADP slowly dissociates, a second ATP binds. The binding correlates with an increase in the ATPase activity. In contrast the tight binding of [3H]GTP to noncatalytic sites gives an enzyme with no ATPase activity. The three noncatalytic sites differ in their binding properties. The noncatalytic site that remains vacant after the ADP-heat-activated CF1 is exposed to Mg2+ and ADP and that can bind ATP rapidly is designated as site A; the site that fills with ATP as ADP dissociates when this enzyme is exposed to Mg2+ and ATP is called site B, and the site to which ADP remains bound is called site C. Procedures are given for attaining CF1 with ADP at sites B and C, with GTP at sites A and/or B, and with ATP at sites A, B, and/or C, and catalytic activities of such preparations are measured. For example, little or no ATPase activity is found unless ATP is at site A, but ADP can remain at site C with no effect on ATPase. Maximal GTPase activity requires ATP at site A but about one-fifth of maximal GTPase is attained when GTP is at sites A and B and ATP at site C. Noncatalytic site occupancy can thus have profound effects on the ATPase and GTPase activities of CF1.     

Independent bindings of Mn2+ and Mg2+ to the active site of B. cereus glutamine synthetase

Glutamine synthetase purified from Bacillus cereus IFO 3131 was modified by iodoacetamide and the ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). Only Mg2+-dependent activity was inactivated by iodoacetamide, whereas only Mn2+-dependent activity was inactivated by FSBA. When iodoacetamide-treated enzyme was reacted with FSBA, Mn2+-dependent activity was also inactivated. Mg2+ plus Mn2+-dependent activity was inactivated in any case. The results suggested that the binding sites of Mn2+ and Mg2+ are separate from each other in the active site of B. cereus glutamine synthetase and that bindings of Mg2+ and Mn2+ to each site are required for normal activity in vivo.