[68] Benzophenone-ATP: A photoaffinity label for the active site of ATPases

[68]

PHOTOAFFINITY LABELINGWITH BzATP

667

[68] B e n z o p h e n o n e - A T P : A P h o t o a f f i n i t y L a b e l for t h e Active Site of A T P a s e s B y NOREEN W I L L I A M S , SHARON H. ACKERMAN, and

PETER S. COLEMAN Introduction I Photoaffinity labels have enjoyed an increasing popularity in enzymology since their introduction by Westheimer and colleagues. 2,3 When successfully designed and applied, these molecular probes seek out selective domains on macromolecular targets for which they possess a substratelike affinity, and upon irradiation with actinic light, form covalent crosslinkages at these specific binding sites. Such an approach can be extremely valuable for enzyme structure/mechanism studies. The site-specific affinity of photoreactive substrate analogs leads to covalent occupancy of the catalytic site of enzymes, which causes irreversible enzyme inhibition and provides a unique opportunity for examining the primary sequence at the active site. Due to the very large number of enzymes requiring adenine nucleotides (especially ATP) as primary or cosubstrate, there has been much interest in designing ATP derivatives that are photochemically active. Most of the available photoaffinity probes contain the azide group (--N3) as the photoactive center. 4-7 While there is no doubt that azido derivatives of ATP (and of other substrates or ligands) have proved very useful, the photochemical properties of the intermediate nitrene species display complicated reaction pathways that are not always easily predictable or understood mechanistically, and thus the azido compounds may offer the experimentalist some undesired difficulties, s-l° 1 The original work on BzATP and rat liver FrATPase was funded, in part, by Biomedical Research Support Group Grant RR07062. 2 F. H. Westheimer, Ann. N.Y. Acad. Sci. 346, 134 0980). 3 V. Chowdry and F. H. Westheimer, Annu. Rev. Biochem. 48, 293 (1979). 4 B. E. Haley and J. Hoffman, Proc. Natl. Acad. Sci. U,S.A. 71, 3367 (1974). 5 H. Bayley and J. R. Knowles, this series, Vol. 46, p. 69. 6 R. J, Guillory and S. J. Jeng, this series, Vol. 46, p. 259. 7 A recent collection of symposium papers that deals with photoaffinity labeling with either carbene or nitrene precursor analogs is given in Fed. Proc., Fed. Am. Soc. Exp. Biol. 42, 2825 (1983). 8 H. Bayley and J. R. Knowles, Biochemistry 17, 2414 (1978). 9 j. V. Staros, Trends Biochem. Sci. 5, 320 (1980). 10 p. V. Vignais, A.-C. Dianoux, G. Klein, G. Lauquin, J. Lunardi, R. Pougeois, and M. Satre, Prog. Clin. Biol. Res, 102B, 439 (1982).

METHODS IN ENZYMOLOGY, VOL. 126

Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

668

REVERSIBLEATP SYNTHASE(FoFj-ATPase)

[68]

In this chapter we present, together with methods, arguments in support of the use of benzophenone as a preferred photoactive moiety in the design of photoaffinity substrate analogs, referring specifically to 3'-O-(4benzoyl)benzoyl-ATP (so-called benzophenone-ATP or BzATP). Here we shall limit discussion to our experience with BzATP and the F1ATPase of rat liver mitochondria, N,J2 which has given rise to more recent work by other laboratories where successful photolabeling has been achieved with BzATP (or BzADP) and different ATPase enzymes.13-J6"16a One of the most attractive aspects of BzATP as a photoaffinity probe, in addition to the unique photochemical properties of benzophenone described below, is the relative ease with which it is synthesized. These factors should help to encourage the use of BzATP (and other "Bzligand" derivatives) as being particularly well suited for studies of ATPutilizing (and other) enzymes. Readers should note, however, that the benzophenone functional group is attached to the adenine nucleotide moiety via a 3'-ester linkage (see Fig. 1). Despite evidence that this ester bond seems to remain stable under mild acid/base conditions jH2 (cf. Characterization of BzATP, pH Stability), recent experience indicates that the bond is labile under the more extreme conditions required by some laboratory protocols, such as those involving protein sequencing. Consequently, the most effective and unambiguous use of benzophenone-derivatized photoaffinity probes probably necessitates methods for the prior synthesis of [4-~4C]carboxyben zophenone, 16,j7 or derivitization of the nucleotide with [3-3H]-4-benzoylbenzoic acid, which has recently become available commercially (Cat. #TNC-455, Rotem Industries Ltd., P. O. Box 9046, Beer Sheva, Israel). Some General Considerations The following discussion is presented in order to clarify the underlying logic pertaining to the use of benzophenone-derivatized ligands for receptor domains on diverse biological macromolecules in an aqueous reaction medium. Formalized treatments of the photochemistry underlying these concepts are available in several excellent monographs. 18-20 zl N. Williams and P. S. Coleman, J. Biol. Chem. 257, 2834 (1982). 12 N. Williams, Ph.D. thesis, New York University, 1981. 13 D. Bar-Zvi, M. Tiefert, and N. Shavit, FEBS Lett. 160, 233 (1983). 14 N. G. Kambouris and G. G. Hammes, Proc. Natl. Acad. Sci. U.S.A. 82, 1950 (1985). t5 M. B. Cable and F. N. Briggs, J. Biol. Chem. 259, 3612 (1984). 16 R. Mahmood and R. G. Yount, J. Biol. Chem. 259, 12956 (1984). 1~ M. F. Manolson, P, A. Rea, and R. J. Poole, J. Biol. Chem. 260, 12273 (1985). 17 D. Licht and P. Coleman, unpublished results (1985); also, K. Nakamaye and R. G. Yount, J. Label. Compnd. Radiopharm. 22, 607 (1985). t8 N. J. Turro, "Modern Molecular Photochemistry." Benjamin/Cummings, Reading, Massachusetts, 1978. 19 D. O. Cowan and R. L. Drisko, "Elements of Organic Photochemistry." Plenum, New York, 1976.

[68]

PHOTOAFFINITYLABELINGWITH BzATP

669

0

N~" N--C--N II ~

N

~ CDI

~dry ( IOMF$

~.

O

O "-~

J ~- C--O--C--N

N

O

0

Bz-imidazolide \OH

-~ N - ~ - NH

ArP( q)

BBA

,.@. NH2

O

O

BzATP FIG. 1. Reaction route for BzATP synthesis.

There appear to be at least three physicochemical considerations that, when satisfied, will determine whether photoaffinity labeling with the benzophenone-ATP (or any other photoaffinity) probe occurs at a ligand binding site on the target enzyme. Such specific site labeling is, of course, the desired outcome with any affinity label, in contrast to a less sitespecific and more random covalent cross-linking. These considerations are as follows: (1) the "residence time" (i.e., the lifetime of the probe : enzyme complex) of the BzATP triplet intermediate at the specific binding site(s) on the enzyme prior to covalent cross-linking; (2) the magnitude of :0 j. G. Calvert and J. N. Pitts, "Photochemistry." Wiley, New York, 1966.

670

REVERSIBLEATP SYNTHASE(FoFi-ATPase)

[68]

the second-order rate constant of hydrogen atom abstraction from the binding site domain by the benzophenone triplet intermediate, compared with the first-order rate constant (i.e., the lifetime -j ) for the decay of the photoinduced triplet; and (3) the use of experimental conditions that guard against the possibility that any as yet unbound BzATP triplets, diffusing away from the ATP binding site, would lead to fortuitous labeling at other domains on the enzyme not directly involved with catalysis. For multimolecular reactions whose mechanisms consist of many intermediate steps, one can argue that the lifetime of the reaction complex (such as an ES transition state complex) is the principal consideration. Clearly, the so-called residence time of the BzATP triplet at the catalytic site of an ATPase would be dominated by the affinity of the probe for that site. First, consider, as an example at one extreme, the dissociation of nonspecific complex AB (AB ~ A + B). If there is no structurally based affinity of A for B, the AB association can display half-times estimated to be as short as 10-13 sec.2122 At the other extreme, the mean lifetimes of ES complexes have been found to be quite durable, between 10-7 and 10-4 sec, indicative of "substrate anchoring" due to a specific affinity of the enzyme for the ligand. 22-27 Therefore, if other physicochemical circumstances required for photochemical cross-linking are optimized (see below), specific site labeling may be achieved, or at least may be heavily favored, by stipulating that the residence time of the BzATP at the ATP binding site of the enzyme manifests a duration in the same general range of otherwise productive E : ATP complexes. A second major concern is the rate constant for the decay of the benzophenone triplet state in degassed H20, which was found to be about 104 sec -1 via flash photolysis spectroscopy. 28 For photoaffinity labeling, one must ask whether such a triplet lifetime is "long" or "short" relative to the rate of its productive reaction via hydrogen abstraction from the target enzyme to be labeled. By means of a flash photolysis experiment in a degassed aqueous system, this laboratory 29 measured a rate constant of 2~ M. Frost and R. Pearson, "Kinetics and Mechanism," Chap. 11. Wiley, New York, 1961. 22 j. Reuben, Proc. Natl. Acad. Sci. U.S.A. 68, 63 (1971). 23 j. T. Gerig, J. Am. Chem. Soc. 90, 2681 (1968). 24 j. T. Gerig and J. D. Reinheimer, J. Am. Chem. Soc. 92, 3147 (1970). 2~ B. D. Sykes, P. D. Schmidt, and G. R. Stark, J. Biol. Chem. 245, 1180 (1970). 26 A. G. Marshall, Biochemistry 7, 2450 (1968). 27 A. S. Mildvan and M. C. Scrutton, Biochemistry 6, 2978 (1967). M. B. Ledger and G. Porter, J. Chem. Soc., Faraday Trans. 1 68, 539 (1972). 29 V. A. Kuzmin and P. S. Coleman, unpublished results (1981). BzATP solutions (pH 7.0) in deionized water were thoroughly degassed on a high-vacuum line by means of the freezepump-thaw method. The flash absorption spectroscopy apparatus was kindly provided by the Department of Chemistry, New York University. J. Navarro assisted with the experiments.

[68]

PHOTOAFFINITY LABELINGWITH BzATP

671

6.4 - 0.8 x 108 M - t sec-i for the dissipation of triplet BzATP, presumably by hydrogen abstraction to yield the ketyl radical, the hydrogen being provided by either another ground or another triplet state BzATP in the environment. The value we obtained is not far from that for the diffusion rate constant of small solute species in water (-109 M-1 sec-1)18 and agrees nicely with rate constants that have been found for hydrogen abstraction by triplet benzophenone from some solvent donors. 3° The important fact is that a comparison of these rate constants indicates that in water, to which the benzophenone triplet is nearly inert, 28 even random collision of solute molecules by diffusion yields a rate of hydrogen abstraction that is more than four orders of magnitude larger than the lifetime of the benzophenone triplet. Since the probability for hydrogen abstraction would be enhanced to an even greater extent by a stabilized spatial proximity between the triplet benzophenone and its target (as found in E : B z A T P complexes), the phenomenon of diffusion is eliminated altogether from the argument. Therefore, with residence times for BzATP and an ATPase on a time scale similar to those estimated for many long-lived ES complexes, we can expect site-specific covalent coupling with the BzATP triplet to be a highly probable and efficient reaction. Regarding conditions that would help preclude fortuitous, random-site labeling, we comment upon the low energy of the benzophenone triplet state (69 kcal/mol), TM which, although ensuring its relative inertness toward reaction with water, 28,31,32 still leaves it vulnerable to collisional triplet-triplet quenching by the molecular oxygen (a ground state triplet)

:6--6: .°

.*

dissolved in our aqueous enzyme assay medium. In air-equilibrated water at room temperature ([02] = 0.1 mM), the rate constant for the oxygen quenching of triplet benzophenone (and by analogy, of any triplet state BzATP "free" in air-equilibrated aqueous solution) is probably close to 4 × 108 M -~ sec-l. 28 However, it seems unlikely that both molecular oxygen and BzATP would occupy, simultaneously, exactly the same location at the specific nucleotide binding site on the ATPase enzyme. Consequently, only that fraction of the BzATP triplet concentration 30 An interesting example is the rate constant (2.5 x 108 M-~ sec -I) for the photoreduction of the benzophenone triplet by a primary amine, such as 2-butylamine [S. G. Cohen, A. Parola, and G. Parsons, Jr., Chem. Rev. 73, 141 (1973)]. 2-Butylamine may be taken as a conceivable structural analog for the 6-NH2 group "vicinity" on the purine ring of ATP, a source of donatable hydrogen atoms for a colliding triplet state benzophenone. For isopropanol as hydrogen-donating solvent, see C. Walling and M. Gibian, J. Am. Chem. Soc. 86, 3902 (1964). 31 A. Beckett and G. Porter, Trans. Faraday Soc. 59, 2038 (1963). 32 V. A. Kuzmin and A. K. Chibisov, Teor. Eksp. Khim. 7, 403 (1971).

672

REVERSIBLEATP SYNTHASE(FoFvATPase)

[68]

which is n o t anchored at the ATP binding site on the enzyme, but is freely diffusing in the aqueous medium, would be susceptible to rapid annihilation to the ground state upon collision with dissolved O2. Consonant with this reasoning, we believe that with an air-equilibrated enzyme assay medium, nonspecific photo-cross-linking at random surface domains of the ATPase enzyme would probably not occur because of the ample presence of molecular oxygen as triplet quencher. On the basis of this argument and our empirical results, we have found it unnecessary to add radical scavenger molecules, as are often employed to eliminate randomsite photo-cross-linking. 33 It is important to note that the benzophenone triplet probably does not undergo intramolecular structural rearrangement) 4 In contrast, arylazido probes readily rearrange from the excited singlet state and react covalently as electrophilic agents only at nucleophilic functional groups on the target e n z y m e ) 5 Furthermore, by insertion into water, the singlet nitrene introduces a " n e w " (chemically inert) species into the system that competes for the substrate-specified target site. When such undesirable side reactions occur with photolabile probe molecules, the yield of covalent incorporation at the desired target site is lowered considerably. 36 Although substantially less information is available regarding the photochemical mechanisms that predominate with benzophenone-linked affinity probes, they appear to be relatively free of such complications. Finally, we wish to emphasize that introduction of a new photoaffinity probe into the arsenal of useful molecular tools employed by the biochemist must await its successful application by other laboratories. In this regard, the apparent usefulness of benzophenone-derivatized photoaffinity probes (particularly Bz-adenine nucleotides for studies on the general category of ATPase enzymes) is supported empirically by the following information. Different laboratories have recently demonstrated that while BzATP may (or may not) act as a substrate, it is unquestionably a good nucleotide-site ligand for at least five distinct ATP-hydrolyzing enzymes: the rat liver Fill; the chloroplast Fll3'14; the Ca2+,Mg2+-ATPase of the sarcoplasmic reticulum~5; the myosin SFrATPaset6; and the tonoplast ATPase of beet root. j6a Recent preliminary evidence also seems to indicate that BzATP may be a substrate for certain phosphotransferases, such as creatine kinase. 37,38 33 A. E. Ruoho, H. Kiefer, P. E. Roeder, and S. J. Singer, Proc. Natl. Acad. Sci. U.S.A. 70, 2567 (1973). 34 The possibility of forming peroxydiradicals in aerated aqueous solution cannot be excluded, however. 35 B. DeGraff, D. Gillespie, and R. Sundberg, J. Am. Chem. Soc. 96, 7491 (1974). 36 p. E. Nielsen and O. Buchard, Photochem. Photobiol. 35, 317 (1982). 37 A. Vinitzky and C. Grubmeyer, Ann. N . Y . Acad. Sci. 435, 222 (1984). 3s It would also appear that the enzyme pyruvate kinase can utilize BzADP as substrate.

[68] Preparation

PHOTOAFFINITY LABELING WITH B z A T P of 3'-O-(4-Benzoyl)benzoyl-ATP

673

( B z A T P ) 39

Synthesis N,N'-Dimethylformamide (DMF; Gold Label, Aldrich) is distilled and then stored o v e r MgSO4, or better, over a molecular sieve (type 4A, 8-12 mesh, Aldrich) in an evacuated desiccator until used. The carboxyl group activator, l,l'-carbonyldiimidazole (CDI), the purity of which must be accounted for in this synthesis (10.46 g, 64.5 mmol, Aldrich), and 4benzoylbenzoic acid (BBA; 4.75 g, 21 mmol, Aldrich) are allowed to react in 25 ml of the anhydrous DMF in a light-shielded, 500-ml round-bottom flask at room temperature, with vigorous stirring. The ensuing reaction thickens to an opaque white mass within 15 min. A 125-ml solution of 0.03 M ATP (disodium salt in deionized water, Sigma, Grade I) is then added slowly with good stirring. 4° The mixture immediately evolves CO2 (Fig. 1), but retains its opaque white appearance for the first hour of reaction. Clearing of the reaction occurs with overnight stirring to yield a yellowish, straw-colored solution. After the reaction has finished, the volume (150 ml) is reduced to about 15 ml by rotary evaporation under vacuum with very mild heating. The crude product is precipitated in the flask with about 200 ml of acetone. The off-white precipitate is repeatedly washed with acetone on Whatman #1 filter paper by B0chner funnel vacuum filtration to remove the majority of unreacted CDI and BBA. The powdered crude product on the filter is dried by vacuum desiccation and stored desiccated at - 2 0 ° . Alternatively, the 15-ml viscous solution is precipitated in the reaction flask with acetone, as described above. Then the flask is cooled on ice, the hygroscopic (somewhat sticky) fine precipitate is allowed to settle, and the acetone is decanted. Fresh acetone (100 ml) is added and the slurry is transferred to 30-ml Corex tubes (Corning) and repeatedly washed by centrifugation (I0,000 g, 5 min, 0°). The acetone supernatant, which con-

This enzyme was found capable of catalyzing the regeneration of the BzATP employed in the F r A T P a s e assay (cf. Ref. 47), but it was necessary to add it to the assay cocktail in excess (9-10 additional units) to preclude the coupled enzyme assay itself from becoming rate limiting for the evaluation of FrATPase activity (see Ref. 11). 39 The abbreviations used are as follows: DMF, N, N'-dimethylformamide; BBA, 4-benzoylbenzoic acid; Bz-, any 4-carboxybenzophenone-derivatized reagent; BzATP, 3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate; CDI, l,l'-carbonyldiimidazole; SMP, sonicated submitochondrial particles. 4o The rationale for using a 1 : 5 DMF : water volume ratio as reaction solvent in this system has been discussed by B. P. Gottikh, A. A. Krayevsky, N. B. Tarussova, P. P. Purygin, and T. L. Tsilevich, Tetrahedron 26, 4419 (1970), and reiterated by R. J. Guillory and S. J. Jeng, this series, Vol. 46, p. 259, as the means for promoting preferential derivatization of the ATP at the ribose hydroxyl functions.

674

REVERSIBLEATP SYNTHASE(FoFj-ATPase)

[68]

tains diminishing amounts of starting materials (Fig. 1, step 1), is discarded. The resulting crude product, which has lost much of its stickiness, is dried in the presence of P205 under vacuum desiccation and stored at - 6 0 ° until used. One of the benefits of the synthesis of BzATP given above is that it is accomplished via one simple reaction setup. There are no stable intermediates to be isolated and characterized en route. Radioactive BzATP (3H and 32p) is synthesized according to the same protocol from commercially obtained ATP labeled with either 3H or 32p.11 Purification o f B z A TP

Originally, H our purification of the crude product was performed on a light-shielded column of Sephadex LH-20 (Pharmacia Fine Chemicals) with a bed volume of 1800 ml. In this method, crude product (150-200 mg) is dissolved in - 5 ml elution buffer (0.1 M ammonium formate, pH 7.4), the flow rate adjusted to 2 ml/min, and the elution profile monitored at 260 nm. The fourth of five peaks (elution volume = 1620 ml) consists of the purified BzATP as determined by TLC analysis (see below). An equally satisfactory, but significantly more rapid procedure employs a reversed-phase "flash" column chromatography method. 41,4zTwo approaches have proved successful. With the first, the stationary phase sorbent consists of octadecyl (C18) chains bonded to silica gel (40 /~m particle size, 60 A pore size, J.T. Baker). A heavy-walled glass column (1.9 cm i.d. × 35.6 cm high) is packed with the dry bead sorbent to a height of 15 cm (42 ml bed volume), and the purified product is resolved by elution with 35% methanol : 65% 0.75 M ammonium formate (by volume), pH 8.5, passed through the column under 20 psi N: pressure at a flow rate of 20 ml/min. Crude product (30-50 mg) is mixed with a minimal volume of water, loaded on the flash chromatography column, and 5-ml fractions are collected by hand (due to the rapidity of the flow rate). The absorbance of the eluate at 260 nm is measured (Fig. 2). The peak fractions containing the purified BzATP, eluting after 250-325 ml (13-17 min), are pooled and lyophilized. Several lyophilizations are performed after redissolving the product in deionized water. In the second method, the stationary phase sorbent is the silicabonded octyl (C8) moiety. Elution is performed discontinuously. First, after crude product ( - 5 0 mg) is loaded onto the column, a solvent containing 30% methanol : 70% 0.175 M ammonium formate (by volume), pH 7.6, is passed through, under 20 psi N2 pressure, and both ATP and BBA 41w. c. Still, M. Kahn, and A. Mitra, J. Org. Chem. 43, 2923 (1978). 4zL. J. Crane, M. Zief, and J. Horvath, Am. Lab. 13, 128 (1981).

[68]

PHOTOAFFINITY LABELINGWITH BzATP

4.00

675

D

.--ATP

3.50

5.00

2.50

o 200 ~2

1.50

1.00

0.50

0

10

20

30

40

50

60

70

80

90

FRACTION NUMBER FIG. 2. " F l a s h " chromatography column elution profile (5-ml fractions) of a typical purification; 31 nag of crude, acetone-precipitated product was loaded onto the column. The actual elution volume for the BzATP peak is sensitive to the column pressure and may vary somewhat. We find that the ratio of the peak elution volumes corresponding to BzATP versus BBA is always between 2.6 and 3.2, despite column pressure fluctuations. Peak identification was made by TLC against standards. See text for further details.

are eluted, in that order, with about 400 ml solvent. After the BBA peak has been fully eluted (absorbance at 260-261 nm approaches the base line), the flow is temporarily interrupted. The original low salt-methanol elution solvent is quickly substituted for one containing no salt, which consists of 40% methanol : 60% water (by volume), with pH held to about 7.5. The N2 pressure is reapplied and the BzATP is eluted in the ensuing fractions totaling 75-100 ml, which are pooled, lyophilized twice, and stored at - 7 0 ° . This method results in the recovery of product essentially as the free acid. With either the LH-20 or the flash column procedure, the yield is usually about 20% relative to the starting amount of ATP. Upon lyophili-

676

REVERSIBLEATP SYNTHASE(FoF1-ATPase)

[68]

zation, the purified BzATP is dried over P205 in a vacuum desiccator and stored at - 7 0 °.

Characterization of BzATP

Thin-Layer Chromatography Two TLC procedures are used. In the original method, microcrystalline TLC plates with fluorescent indicator (Avicel, Analtech) are developed with 1-butanol/acetic acid/HzO (5 : 1 : 3 by volume). The Rf values obtained are ATP, 0.12; B zATP, 0.63; benzoylbenzoic acid, 0.81. In the second procedure, reversed-phase TLC on 1 × 3 in. MKC18 F plates (Whatman, 200/zm thickness) are developed with a buffer containing 55% methanol : 45% 0.75 M ammonium formate (by volume), pH 8.5. Analysis of the crude product with this reversed-phase system reveals three spots: B z A T P ( g f ~-- 0.16); benzoylbenzoic acid (Rf = 0.30); and unreacted ATP ( R f = 0.62). Occasionally, a fourth spot, Rf = 0.58, indicative of a small amount of ATP dephosphorylation to ADP, may be observed. If, prior to developing the TLC plate (with either procedure), the BzATP at the spotting origin is wetted with water and exposed for a few minutes to longwavelength irradiation from a UVSL-25 mineralight (Ultraviolet Products), a substantial portion of the spotted material remains immobilized at the origin subsequent to development, with no evidence of breakdown products observed. This provides preliminary assurance that the BzATP product is covalently photoreactive. We generally run TLC analyses in pairs: one plate having been exposed to actinic UV light as indicated, and the other run normally for verification of Rf values.

pH Stability of BzATP Purified BzATP (0.01 M) was incubated for 30 min (25 °) over a broad pH range, 4-10, in 10 mM Tris-maleate. Subsequent TLC analysis, with ATP and 4-benzoylbenzoic acid as standards, showed no evidence of ester hydrolysis.

Elemental Analysis Commercial elemental analysis of the purified BzATP (as the ammonium salt) confirmed a unit stoichiometric addition of the benzophenone moiety to each mole of ATP in the final product. The results were as follows: Calculated: C 36.60; H 4.36; N 12.45; P 12.97; Experimentally determined: C 36.30; H 5.10; N 12.40; P 12.63.

[68]

PHOTOAFFINITYLABELINGWITH BzATP

677

Proton NMR Proton N M R spectra 12 of purified BzATP in D20 indicate two regions of interest, each of which corresponds to the NMR profile of pure ATP and benzoylbenzoic acid, respectively. One region is identifiable with exchangeable protons on the ribosyl substituent of ATP between about 4 and 6 ppm. A second (downfield) region between 7.1 and 8.5 ppm corresponds to exchangeable protons affiliated with aromatic (benzophenone and purine) substituents. All resonance peaks can be accounted for by comparison with ATP and benzoylbenzoic acid, except for a chemical shift from about 4.3 ppm to nearly 6.25 ppm, observed with BzATP but not with ATP, which is attributable to a substitution at the 3' position of the ribose moiety. 6 UV Absorbance T h e )kmax for BzATP in phosphate buffer, pH 7.0, is 261.5 nm. As indicated earlier, we have also found it possible to purify the BzATP product from the flash chromatography column under low- to no-salt conditions by employing a discontinuous elution solvent protocol. The BzATP thus obtained, upon lyophilization, may be taken to be fully protonated and assumed to contain 2 mol of bound H20 (MW = 751). In 10 mM KH~PO4 buffer, pH 7.0, the eM at 261 nm for the purified, salt-free BzATP was determined to be 3.192 -+ 0.137 × 104 M -~ × cm -~, the value we use in our enzyme studies. It should be noted, however, that purification of BzATP in high salt (0.75 M ammonium formate/methanol, see Fig. 2) seems to help preserve the stability of the lyophilized product upon storage.

General Remarks It is difficult to state unequivocally that BzATP exists mainly as the 3'O-substituted derivative, but the evidence at hand appears to indicate that this is the case. Although the 2'-hydroxyl is probably preferred as the kineticiaUy favored esterification site, the 3'-substituted isomer is thermodynamically more stable, and it is reasonable to conclude that acyl migration occurs during the synthetic reaction. 43,44A small amount of 2'-isomer contamination would probably not be detectable by our analytical measurements, and indeed, for experiments usually performed with BzATP, the adenine ring system and phosphate groups are the more significant 43 p. G. Zamecnik, Biochem. J. 85, 257 (1962). C. S. McLaughlin and V. M. Ingrain, Biochemistry 4, 1442, 1448 (1965).

678

REVEgStBLEATP SVNTHASE(FoF~-ATPase) 0 II

[68]

0° II h~

(~340 nrn)

a-[~

0_

0

0

O--H I

0°

II

c (a} [ ~ " - O - C 0

n,~*isO) (dirodicoltriplet)

F~-[CHa]-R

+ F,-[~H]-R

(hydrogenObstroctiOn)~"[ ~ " - - 0 - - ~ 0 t

~

,ATPoseradico,] I

ICl - [CH]- R

0

(eovolentodduct )

FIG. 3. Probable reaction pathway for covalent photoaffinitylabeling of FrATPase by BzATP. elements correlating with affinity of the probe/substrate for the ATPutilizing enzyme. Photolabeling ATPase Enzymes with BzATP Mechanism The n --~ It* absorption band for BzATP has a wavelength maximum at about 350 nm in water, with a very weak extinction coefficient (-< 160 M -1 cm -~) characteristic of this type of electronic transition.18 Upon irradiation with wavelengths >340 nm, the excited singlet state of benzophenone rapidly (-~10 -~ sec) undergoes intersystem crossing to the metastable triplet state with nearly 100% efficiency. This triplet state posseses diradicaloid characteristics and may undergo productive photolabeling by means of the postulated two-step sequence given in Fig. 3: (1) hydrogen atom abstraction from the target molecule to yield a ketyl radical of the benzophenone plus a target molecule free-radical species; (2) radicalradical coupling of the ketyl and target species intermediates to generate a covalent C - - C bond between the components. The requirement for irradiation of benzophenone derivatives at about 350 nm (-<82 kcal/mol) nearly eliminates the potential for photodestruction of proteins, which may occur at shorter wavelengths.

[68]

PHOTOAFFINITY LABELINGWITH BzATP

679

Methods

For our early work, llA2 we used the hand-held, low-intensity UVSL25 mineralight (Ultraviolet Products), employing the long-wavelength setting. A Pyrex filter placed over the illuminating surface ensures transmission of only wavelengths above 300 nm. The low-intensity radiation emission provided by this lamp proved sufficient to yield good photoincorporation of the BzATP probe, obviating the requirement for a more elaborate apparatus. However, an alternative and more efficient irradiation setup is provided by a high-pressure HBO 200 W mercury arc lamp (Osram), with Universal lamp housing and a model MTr 14 power supply (Wild-Heerbrugg). This apparatus possesses a very high-intensity light source, especially around 334 and 366 nm, and is equipped with an iris/ diaphragm that can be closed down to irradiate a relatively small area (about 1 cm in diameter at a distance of 30 cm), allowing for shorter sample irradiation times (rarely exceeding 1 rain). It should be emphasized, however, that under appropriate conditions, either of these light sources yields good photoincorporation of BzATP. Our photolysis incubations with purified rat liver FI-ATPase and sonicated submitochondrial particle (SMP) preparations were performed in 1cm, 3-ml quartz fluorescence cuvettes, positioned 5 cm from the UVSL25 mineralight. The effector molecule (BzATP, ATP, or BBA) was added to the cuvette 30 sec prior to irradiation. Photolysis time was 10 min or less. With SMP (1.25 mg/ml), incubations were performed at 10° during photolysis, and after irradiation the particles were sedimented at 140,000 g (45 min). The pellet was rinsed twice with cold deionized water to remove unbound ligand, then resuspended in cold deionized water to 20 mg/ml and assayed for ATPase activity. With the soluble FI-ATPase, the temperature was maintained at 25 ° during irradiation, after which the reaction was dialyzed against 250 mM potassium phosphate, 5 mM EDTA, pH 7.5, to remove unbound ligand. The dialysate was concentrated by application of dry Sephadex G-25 to the outside of the dialysis tubing before being assayed for ATPase activity. SMP preparations and SMP-ATPase assays were accomplished via the methods of Kaplan and Coleman. 45 Rat liver mitochondrial FI was prepared essentially according to Catterall and Pedersen. 46 The isolation medium prior to sonication contained 4 mM ATP. Following the concentration of the FI enzyme after purification, it was lyophilized in 250 mM potassium phosphate, 5 mM EDTA, pH 7.5, and stored at - 6 0 °. F145R. S. Kaplan and P. S. Coleman, Biochim. Biophys. Acta 501, 269 (1978). W. A. Catterall and P. L. Pedersen, J. Biol. Chem. 246, 4987 (1971).

680

REVERSIBLE ATP SYNTHASE (FoFrATPase)

K I N E T I C C O N S T A N T S FOR THE

TABLE 1 ATPase OF SMP

[68]

A N D F I IN T H E A B S E N C E

OF ILLUMINATION"

System

Substrate

K~ (mM)

Vma×(/xmol/min/mg)

KIADP(mM)

SMP

ATP BzATP ATP BzATP

0.16 0.13 0.83 0.94

3.20 0.36 20.71 2.51

0.61 0.07 0.36 0.06

FI

" With SMP, medium contained 10 mM Tris-maleate (pH 7.2), 1.0 mM MgC12, ATP or BzATP (0.05-1.0 mM), ADP (0.05-1.0 mM). When ATP was substrate, incubations contained 0.50 mg SMP protein/ml. Reactions were run for 10 sec at 28° and terminated with 6% (v/v) perchloric acid. With isolated F1, the assay was performed essentially as described according to Ref. 48 with ATP or BzATP concentrations = 0.3-2.0 raM. When ATP was substrate, incubations contained 1.5 /zg Fj protein/ml. When BzATP was substrate, incubations contained 3.0/~g Ft protein/ml plus an additional 9 units of pyruvate kinase. Assays for competition studies were performed essentially as described in Ref. 47 in a total volume of 1.0 ml that contained 50 mM Tris-HC1 (pH 7.5), varying concentrations of both ADP and either ATP or BzATP, a MgCl2 concentration equal to total adenine nucleotide concentration, and 9 p.g purified F~-ATPase. Reactions were run at room temperature for 2 min. A T P a s e a c t i v i t y w a s m e a s u r e d with an A T P r e g e n e r a t i n g a s s a y s y s t e m a c c o r d i n g to e s t a b l i s h e d p r o c e d u r e s . 46,47

R e s u l t s with R a t L i v e r M i t o c h o n d r i a l F r A T P a s e jj B z A T P as S u b s t r a t e

S t u d i e s w i t h b o t h S M P - A T P a s e a n d t h e s o l u b l e F1 e s t a b l i s h e d B z A T P a s a f u n c t i o n a l s u b s t r a t e f o r t h e A T P a s e e n z y m e c o m p l e x in t h e a b s e n c e o f a c t i n i c i l l u m i n a t i o n ( T a b l e I). T h e a p p a r e n t Km v a l u e s for B z A T P relat i v e to A T P a r e i d e n t i c a l w i t h e i t h e r t h e m e m b r a n e - b o u n d o r s o l u b l e e n z y m e . S u c h r e s u l t s m a y i m p l y a s i m i l a r b i n d i n g affinity f o r b o t h s u b s t r a t e s at t h e c a t a l y t i c site(s). T h e Vmax w i t h B z A T P as s u b s t r a t e f o r b o t h e n z y m e p r e p a r a t i o n s , a l t h o u g h s u b s t a n t i a l l y d e c r e a s e d c o m p a r e d to c o n t r o l A T P a s e a c t i v i t y , is still 1 1 - 1 2 % t h a t w i t h A T P . T h e r a t e o f h y d r o l y s i s o f b o t h A T P a n d B z A T P (to A D P a n d B z A D P , r e s p e c t i v e l y ) is e f f e c t i v e l y i n h i b i t e d b y A D P w i t h b o t h t h e S M P a n d s o l u b l e A T P a s e ; h o w e v e r , diff e r e n t KI v a l u e s f o r A D P w e r e o b t a i n e d , d e p e n d i n g on t h e u s e o f A T P o r 47 M. E. Pullman, H. S. Penefsky, A. Datta, and E. Racker, J. Biol. Chem. 235, 3322 (1960).

[68]

PHOTOAFFINITY LABELING WITH B z A T P

681

TABLE 11 CONTROLS FOR BzATP PHOTOLABELING WITH SMP-ATPase AND F rATPase" Conditions Illumination 1. 2. 3. 4.

No Yes Yes Yes

5. Yes 6. Yes

% ATPase activity remaining

System variable

SMP-ATPase

Ft-ATPase

4/~mol BzATP/mg 4/zmol ATP/mg 2.5% (v/v) ethanol 4/~mol BBA/mg in 2.5% (v/v) ethanol 4 ~mol BzATP/mg plus 4/zmol ATP/mg 4/zmol BzATP/mg

99.8 95.7 97.0 94.2

98.1 95.6 93.2 91.0

--

91.0

23.0

34.1

a Experiments were performed according to the methods given in the text and Ref. 11. Photolysis time was always 10 rain (UVSL 25 lamp).

BzATP as substrate. The latter is an interesting result that merits further investigation, for it may comment, indirectly, upon mechanistic restrictions that apply to ATP hydrolysis by this ATPase.

Photoinactivation of ATPase by BzATP Photoinactivation experiments require that stringent controls be performed to demonstrate unequivocally that a loss in enzyme activity occurs as a direct result of specific photoincorporation of the analog at the TABLE lII INHIBITION OF MITOCHONDRIAL ATPase BY PHOTOAFFINITY LABELING WITH BzATP" SMP-ATPase

FI-ATPase

Photolysis conditions

% Inhibition

KmATp ( m M )

% Inhibition

KmATp (mM)

1. ATP plus Mgz+ 2. BzATP minus Mgz+ 3. BzATP plus Mg2+

0 51 77

0.16 0.13 0.12

0 -70

0.83 -0.84

For SMP experiments (1.25 mg protein), the BzATP concentration was 4.0/~mol/mg SMP protein. Photolysis was performed for 10 rain at 10° in 1.0 ml. ATP concentration range for SMP-ATPase assay subsequent to photolysis was 0.3-1.0 mM. For soluble F~ experiments (0.15 mg protein), the BzATP concentration was again 4.0/~mol/mg F~ protein. Photolysis conditions were identical to those with SMP, except the temperature was 25°. The ATP concentration range for the FrATPase assay subsequent to photolysis was 0.3-2.0 mM. See text and Williams and Coleman ~' for further details.

682

REVERSIBLEATP SYNTHASE(FoFI-ATPase)

[69]

ATPase catalytic site. All other irrelevant processes, such as nonspecific labeling, interference by any unanticipated photogenerated intermediates different from BzATP, and photodestruction of the protein, must be ruled out as the mechanism behind the observed effect. The results of these control experiments are shown in Table II. Both the SMP and soluble F~ATPase, irradiated in the absence of effector, indicate little loss in activity. No evidence was obtained for the ability of BzATP to form an inhibitory complex in the dark with either membrane-bound or soluble enzyme, inasmuch as no loss of ATPase activity accompanied such incubations. When either SMP or soluble enzyme was irradiated with only the photolabile moiety of the BzATP, i.e., 4-benzoylbenzoic acid (BBA), virtually no loss in subsequently assayed ATPase activity was observed. This is an important finding, for it demonstrates that the triplet benzophenone itself is not site-specifically directed with respect to the enzyme. Therefore, only a photochemical reaction at the ATP binding site of the enzyme (and not elsewhere) is expected to be inhibitory. A substrate protection experiment conclusively supports this interpretation (Table II); the presence of equimolar ATP and BzATP together during irradiation of the enzyme leads to a >90% retention of ATPase activity. Table III summarizes the following results. Irradiation of SMP with BzATP, without added Mg 2÷, yielded a 51% decrease in the subsequently assayed Vmaxof the ATPase, and a 77% decrease in the Vmaxwhen Mg 2÷ was present during photolysis. Soluble F~, irradiated with BzATP, followed by removal of unbound ligand from the system (via dialysis) and kinetics assays over a range of ATP concentrations, showed an unaltered Km despite a Vmaxdramatically reduced by 70%. Collectively, these data support the catalytic site selectivity of BzATP on the rat liver mitochondrial ATPase complex, whether the enzyme is membrane bound or soluble.

[69] U s e o f A D P A n a l o g s for F u n c t i o n a l a n d S t r u c t u r a l Analysis of Ft-ATPase

By GONTER SCH)~FER, UWE L0CKEN, and MATHIAS LOBBEN General Considerations

The use of substrate analogs which can bind without undergoing catalytic conversion or can be used as covalent markers or reporter molecules at the catalytic domains of proteins is a classical approach to enzyme METHODS IN ENZYMOLOGY, VOL. 126

Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.