Evidence for three alpha subunits in one molecule of F1-ATPase from thermophilic bacterium PS3.

The numbers of sulfhydryl residues in F₁-ATPase of thermophilic bacterium PS3 and its isolated subunits were analyzed with Ellman's reagent. This F₁-ATPase contained three sulfhydryl residues and no disulfide bridge. Of the five kinds of subunits of the F₁-ATPase, only the α subunit contained one sulfhydryl residue. So there are three α subunits in one molecule of the F₁-ATPase.     

Purification by dye-ligand chromatography and a crystallization study of the F1-ATPase and its major subunits, beta and alpha, from a thermophilic bacterium, PS3.

For a crystallization study, purification methods for F1-ATPase from a thermophilic bacterium, PS3, and its major subunits, beta and alpha, have been improved. The improvement depended on the introduction of dye-ligand chromatography columns to the previously adopted array of chromatography columns: a Blue-B (a blue dye bound to agarose) column was introduced for the F1 preparation, a Green-A column (a green dye attached to agarose) for the beta subunit, and a Blue-A (another blue dye, Cibacron Blue 3GA, bound to agarose) column for the alpha subunit. The improved preparations of all the proteins had purities of nearly 99%. Using the highly purified preparations of the proteins, crystallization conditions were searched for in a systematic way. Large plate crystals (0.2 X 0.5 X 0.5 mm) of F1 were grown from a polyethylene glycol solution. However, neither of the subunits was crystallized, in spite of extensive search for crystallization conditions.     

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

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

The (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 has altered ATPase activity after cross-linking alpha and beta subunits at noncatalytic site interfaces

In crystal structures of bovine MF(1), the side chains of alpha F(357) and beta R(372) are near the adenines of nucleotides bound to noncatalytic sites. To determine if during catalysis these side chains must pass through the different arrangements in which they are present in crystal structures, the catalytic properties of the (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex of the TF(1)-ATPase were characterized before and after cross-linking the introduced cysteines with CuCl(2). The unmodified mutant enzyme hydrolyzes MgATP at 50% the rate exhibited by wild type. Detailed comparison of the catalytic properties of the double mutant enzyme before and after cross-linking with those of the wild-type subcomplex revealed the following. Before cross-linking, the (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex has less tendency than wild type to release inhibitory MgADP entrapped in a catalytic site during turnover when MgATP binds to noncatalytic sites. Following cross-linking, ATPase activity is reduced 5-fold, and inhibitory MgADP entrapped in a catalytic site during turnover does not release under conditions wherein binding of ATP to noncatalytic sites of the wild-type enzyme promotes release of MgADP from the affected catalytic site. When assayed in the presence of lauryldimethylamine oxide, which prevents turnover-dependent entrapment of inhibitory MgADP in a catalytic site, ATPase activity of the cross-linked form is 47% that of the unmodified mutant enzyme. These results suggest that, during catalysis, the side chains of alpha F(357) and beta R(372) do not pass through the extremely different relative positions in which they exist at the three noncatalytic site interfaces in crystal structures.     

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

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

The ATPase activity of the alpha 3 beta 3 complex of the F1-ATPase of the thermophilic bacterium PS3 is inactivated on modification of tyrosine 307 in a single beta subunit by 7-chloro-4-nitrobenzofurazan

The catalytically active alpha 3 beta 3 complex, assembled as described (Miwa, K., and Yoshida, M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6484-6487) from the isolated alpha and beta subunits of the F1-ATPase of the thermophilic bacterium PS3 (TF1), is inactivated by 7-chloro-4-nitrobenzofurazan (Nbf-Cl) with characteristics very similar to those observed when TF1, which has the subunit composition, alpha 3 beta 3 gamma delta epsilon, is inactivated by the reagent under the same conditions. Both native TF1 and the alpha 3 beta 3 complex are inactivated by 200 microM Nbf-Cl with a pseudo-first order rate constant of 3.7 x 10(-2) min-1 in the presence of 0.2 M Na2SO4 at pH 7.6 and 23 degrees C. The rate of increase in absorbance at 385 nm of reaction mixtures containing 200 microM [14C]Nbf-Cl and TF1, the wild-type alpha 3 beta 3 complex, or the mutant alpha 3(beta Y307----F)3 complex, each at 18 microM was also examined. Since the alpha 3(beta y307----F)3 complex is resistant to inactivation by Nbf-Cl, difference spectrophotometry revealed that inactivation of native TF1 and the wild-type alpha 3 beta 3 complex could be correlated with formation of about 1 mol of Nbf-O-Tyr/mol of enzyme or complex. Fractionation of peptic digests of the labeled enzyme and complexes by reversed-phase high performance liquid chromatography resolved a major radioactive peptide that was common to labeled TF1 and the labeled alpha 3 beta 3 complex but was absent in the digest of the labeled alpha 3(beta Y307----F)3 complex. This labeled peptide was shown to contain Tyr-beta 307 derivatized with [14C]Nbf-Cl by automatic amino acid sequence analyses. From these results, it is concluded that one-third of the sites' reactivity of Nbf-Cl with Tyr-beta 307 in TF1 or its equivalent in other F1-ATPases is not influenced by the presence of the gamma, delta, or epsilon subunits. It has also been shown that Tyr-307 is not modified to an appreciable extent when the isolated beta subunit is treated with [14C]Nbf-Cl under conditions in which this residue is nearly completely labeled in a single beta subunit when TF1 or the alpha 3 beta 3 complex is inactivated by the reagent.     

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

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

Hydrogen bonds between the alpha and beta subunits of the F1-ATPase allow communication between the catalytic site and the interface of the beta catch loop and the gamma subunit

F(1)-ATPase mutations in Escherichia coli that changed the strength of hydrogen bonds between the alpha and beta subunits in a location that links the catalytic site to the interface between the beta catch loop and the gamma subunit were examined. Loss of the ability to form the hydrogen bonds involving alphaS337, betaD301, and alphaD335 lowered the k(cat) of ATPase and decreased its susceptibility to Mg(2+)-ADP-AlF(n) inhibition, while mutations that maintain or strengthen these bonds increased the susceptibility to Mg(2+)-ADP-AlF(n) inhibition and lowered the k(cat) of ATPase. These data suggest that hydrogen bonds connecting alphaS337 to betaD301 and betaR323 and connecting alphaD335 to alphaS337 are important to transition state stabilization and catalytic function that may result from the proper alignment of catalytic site residues betaR182 and alphaR376 through the VISIT sequence (alpha344-348). Mutations betaD301E, betaR323K, and alphaR282Q changed the rate-limiting step of the reaction as determined by an isokinetic plot. Hydrophobic mutations of betaR323 decreased the susceptibility to Mg(2+)-ADP-AlF(n)() inhibition and lowered the number of interactions required in the rate-limiting step yet did not affect the k(cat) of ATPase, suggesting that betaR323 is important to transition state formation. The decreased rate of ATP synthase-dependent growth and decreased level of lactate-dependent quenching observed with alphaD335, betaD301, and alphaE283 mutations suggest that these residues may be important to the formation of an alternative set of hydrogen bonds at the interface of the alpha and beta subunits that permits the release of intersubunit bonds upon the binding of ATP, allowing gamma rotation in the escapement mechanism.     

Availability to monoclonal antibodies of antigenic sites of the alpha and beta subunits in active, denatured or membrane-bound mitochondrial F1-ATPase

The binding of five monoclonal antibodies to mitochondrial F1-ATPase has been studied. Competition experiments between monoclonal antibodies demonstrate that these antibodies recognize four different antigenic sites and provide information on the proximity of these sites. The accessibility of the epitopes has been compared for F1 integrated in the mitochondrial membrane, for purified beta-subunit and for purified F1 maintained in its active form by the presence of nucleotides or inactivated either by dilution in the absence of ATP or by urea treatment. The three anti-beta monoclonal antibodies bound more easily to the beta-subunit than to active F1, and recognized equally active F1 and F1 integrated in the membrane, indicating that their antigenic sites are partly buried similarly in purified or membrane-bound F1 and better exposed in the isolated beta-subunit. In addition, unfolding F1 by urea strongly increased the binding of one anti-beta monoclonal antibody (14 D5) indicating that this domain is at least partly shielded inside the beta-subunit. One anti-alpha monoclonal antibody (20 D6) bound poorly to F1 integrated in the membrane, while the other (7 B3) had a higher affinity for F1 integrated in the membrane than for soluble F1. Therefore, 20 D6 recognizes an epitope of the alpha-subunit buried inside F1 integrated in the membrane, while 7 B3 binds to a domain of the alpha-subunit well exposed at the surface of the inner face of the mitochondrial membrane.     

The use of dithionite reduction to identify the essential tyrosine residue in the F1-ATPase from the thermophilic bacterium, PS3, that reacts with 7-chloro-4-nitrobenzofurazan

When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by greater than 90% with 7-chloro-4-nitro[14C]benzofurazan ([14C]Nbf-Cl) at pH 7.4, 1.4 mol of [14C]Nbf were incorporated per mol of enzyme. After pepsin digestion of the labeled enzyme at pH 3.0, a single, major peak of radioactivity was resolved by reversed-phase high-performance liquid chromatography under acidic conditions were peptidyl Nbf-O-tyrosine is stable. This radioactive peak, designated RP-1, eluted with a retention time of 95 min. When the material in RP-1 was subjected to reversed-phase high-performance liquid chromatography under the same conditions after treatment with sodium dithionite, a single, major peak of radioactivity, designated RP-2, was resolved with a retention time of 52 min. Automatic Edman degradation of this material revealed that it has the amino acid sequence I-Y*-V-P-A-D-(D), where Y* presumably represents peptidyl [14C]Nbf-O-tyrosine. These results provide the basis for a facile method to purify peptides containing [14C]Nbf-O-tyrosine in which the labeled residues can be identified by amino acid sequence analysis using the Edman degradation.     

Reconstitution of ATPase activity from the isolated alpha, beta, and gamma subunits of the coupling factor, F1, of Escherichia coli.

The three major subunits (α, β and γ) of the coupling factor, F₁ ATPase, of $\text{Escherichia coli}$ were separated and purified by hydrophobic column chromatography after the enzyme was dissociated by cold inactivation. The ability to hydrolyze ATP was reconstituted by dialyzing the mixture of subunits against 0.05 M Tris-succinate, pH 6.0, containing 2 mM ATP and 2 mM MgCl₂. A mixture containing α, β and γ regained ATP hydrolyzing activity. Individual subunits alone or mixtures of any two subunits did not develop ATPase activity, except for a low but significant activity with α plus β. The reconstituted ATPase had a K_m of 0.23 mM for ATP and a molecular weight by sucrose gradient density centrifugation of about 280,000.     

The fluorescence spectrum of the introduced tryptophans in the alpha 3(beta F155W)3gamma subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 cannot be used to distinguish between the number of nucleoside di- and triphosphates bound to catalytic sites.

It has been reported that shifts in the fluorescence emission spectrum of the introduced tryptophans in the betaF155W mutant of Escherichia coli F(1) (bovine heart mitochondria F(1) residue number) can quantitatively distinguish between the number of catalytic sites occupied with ADP and ATP during steady-state ATP hydrolysis (Weber, J., Bowman, C., and Senior, A. E. (1996) J. Biol. Chem. 271, 18711--18718). In contrast, addition of MgADP, Mg-5'-adenylyl beta,gamma-imidophosphate (MgAMP-PNP), and MgATP in 1:1 ratios to the alpha(3)(betaF155W)(3)gamma subcomplex of thermophilic Bacillus PS3 F(1) (TF(1)) induced nearly identical blue shifts in the fluorescence emission maximum that was accompanied by quenching. Addition of 2 mm MgADP induced a slightly greater blue shift and a slight increase in intensity over those observed with 1:1 MgADP. However, addition of 2 mm MgAMP-PNP or MgATP induced a much greater blue shift and substantially enhanced fluorescence intensity over those observed in the presence of stoichiometric MgADP or MgAMP-PNP. It is clear from these results that the fluorescence spectrum of the introduced tryptophans in the betaF155W mutant of TF(1) does not respond in regular increments at any wavelength as catalytic sites are filled with nucleotides. The fluorescence spectrum observed after entrapping MgADP-fluoroaluminate complexes in two catalytic sites of the betaF155W subcomplex indicates that the fluorescence emission spectrum of the enzyme is maximally perturbed when nucleotides are bound to two catalytic sites. This finding is consistent with accumulating evidence suggesting that only two beta subunits in the alpha(3)beta(3)gamma subcomplex of TF(1) can simultaneously exist in the completely closed conformation.     

Single site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP by the F1-ATPase from thermophilic bacterium PS3 is accelerated by the chase-addition of excess ATP.

The interaction of 2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP) and TNP-ADP to F1-ATPase from a thermophilic bacterium PS3 (TF1) was investigated. When TNP-ADP or TNP-ATP was added to the isolated alpha or beta subunit of TF1, characteristic difference spectra were generated for each subunit. Difference spectra generated on addition of these analogs to TF1 resembled those observed for the beta subunit, indicating TNP analogs bind to the beta subunits in the molecule of TF1. Results of equilibrium dialysis showed that TNP-ADP binds to a single high affinity site on TF1 in the presence of Mg2+ with a dissociation constant of 2.2 nM. When TNP-ATP was added to TF1 in a substoichiometric molar ratio, it rapidly bound to TF1 and was slowly hydrolyzed. The hydrolysis proceeded nearly to completion without showing stable equilibrium between bound species of TNP-ATP and TNP-ADP. Similar to beef heart mitochondrial F1, this hydrolysis was greatly accelerated by the chase-addition of 100 microM ATP. However, the hydrolyzed product, TNP-ADP, remained bound on the beta subunit even after the chase.     

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

We performed kinetic studies on the reactions of a fluorescent ATP analog, 2'-(5-dimethyl-aminonaphthalene-1-sulfonyl) amino-2'-deoxyATP (DNS-ATP), with E. coli F1-ATPase (EF1) and its subunits, to clarify the role of each subunit in the ATPase reaction. The following results were obtained. 1. One mol of EF1, which contains nonexchangeable 2 mol ATP and 0.5 mol ADP, binds 3 mol of DNS-ATP. The apparent dissociation constant, in the presence of Mg2+, was 0.23 microM. Upon binding, the fluorescence intensity of DNS-ATP at 520 nm increased exponentially with t1/2 of 35 s, and reached 3.5 times the original fluorescence level. Following the fluorescence increase, DNS-ATP was hydrolyzed, and the fluorescence intensity maintained its enhanced level. 2. The addition of an excess of ATP over the EF1-DNS-nucleotide complex, in the presence of Mg2+, decreased the fluorescence intensity rapidly, indicating the acceleration of DNS-nucleotide release from EF1. ADP and GTP also decreased the fluorescence intensity. 3. DCCD markedly inhibited the accelerating effect of ATP on DNS-nucleotide release from EF1 and the EF1-DNS-ATPase or -ATPase activity in a steady state. On the other hand, DCCD only slightly inhibited the fluorescence increase of DNS-ATP, due to its binding to EF1, and the rate of single cleavage of 1 mol of DNS-ATP per mol of alpha subunit of EF1. 4. In the presence of Mg2+, 0.65-0.82 mol of DNS-ATP binds to 1 mol of the isolated alpha subunit of EF1 with an apparent dissociation constant of 0.06-0.07 microM. Upon binding, the fluorescence intensity of DNS-ATP at 520 nm increased 1.55 fold very rapidly (t1/2 less than 1 s). No hydrolysis of DNS-ATP was observed upon the addition of the isolated alpha subunit. The fluorescence intensity of DNS-ATP was unaffected by the addition of the isolated beta subunit. DNS-ATP was also unhydrolyzed by the isolated beta subunit. 5. EF1-ATPase was reconstituted from alpha, beta, and gamma subunits in the presence of Mg2+ and ATP. The kinetic properties of the fluorescence change of DNS-ATP in the reaction with the reconstituted EF1-ATPase were quite similar to those of native EF1. Most of our findings are consistent with a simple mechanism that the high affinity catalytic site and low affinity regulatory site exist in the alpha subunit and beta subunit, respectively. However, the findings mentioned in (4) suggest that the binding of the alpha and beta subunit, which is mediated by the gamma subunit, induces conformational change(s) in the ATP binding site located probably in the alpha subunit, and that the conformational change(s) is essential to exert the full hydrolyzing activity.     

Comparative Mg(2+)-dependent sequential covalent binding stoichiometries of 3'-O-(4-benzoyl)benzoyl adenosine 5'-diphosphate of MF1, TF1, and the alpha 3 beta 3 core complex of TF1. The binding change motif is independent of the F1 gamma delta epsilon subunits.

Three F1 preparations, the beef heart (MF1) and thermophilic bacterium (TF1) holoenzymes, and the alpha 3 beta 3 "core" complex of TF1 reconstituted from individually expressed alpha and beta subunits, were compared as to their kinetic and binding stoichiometric responses to covalent photoaffinity labeling with BzATP and BzADP (+/- Mg2+). Each enzyme displayed an enhanced pseudo-first order rate of photoinhibition and one-third of the sites covalent binding to a catalytic site for full inhibition, plus, but not minus Mg2+. Titration of near stoichiometric [MgBzADP]/[F1] ratios during photolysis disclosed two sequential covalent binding patterns for each enzyme; a high affinity binding corresponding to unistoichiometric covalent association concomitant with enzyme inhibition, followed by a low affinity multisite-saturating covalent association. Thus, in the absence of the structural asymmetry inducing gamma delta epsilon subunits of the holoenzyme, the sequential binding of nucleotide at putative catalytic sites on the alpha 3 beta 3 complex of any F1 appears sufficient to effect binding affinity changes. With MF1, final covalent saturation of BzADP-accessible sites was achieved with 2 mol of BzADP/mol of enzyme, but with TF1 or its alpha 3 beta 3 complex, saturation required 3 mol of BzADP/mol of enzyme. Such differential final labeling stoichiometries could arise because of the endogenous presence of 1 nucleotide already bound to one of the 3 potential catalytic sites on normally prepared MF1, whereas TF1, possessing no endogenous nucleotide, has 3 vacant BzADP-accessible sites. Kinetics measurements revealed that regardless of the incremental extent of inhibition of the TF1 holoenzyme by BzADP during photolysis, the two higher apparent Km values (approximately 1.5 x 10(-4) and approximately 10(-3) M, respectively) of the progressively inactivated incubation are unchanged relative to fully unmodified enzyme. As reported for BzATP (or BzADP) and MF1 (Ackerman, S.H., Grubmeyer, C., and Coleman, P.S. (1987) J. Biol. Chem. 262, 13765-13772), this supports the fact that the photocovalent inhibition of F1 is a one-hit one-kill phenomenon. Isoelectric focusing gels revealed that [3H]BzADP covalently modifies both TF1 and MF1 exclusively on the beta subunit, whether or not Mg2+ is present. A single 19-residue [3H]BzADP-labeled peptide was resolved from a tryptic digest of MF1, and this peptide corresponded with the one believed to contain at least a portion of the beta subunit catalytic site domain (i.e. beta Ala-338----beta Arg-356).