[53] Labeling Acyl-CoA binding sites with photolabile analogs

[53]

LABELINGACYL-COABINDINGSITES

633

phorylation of PSs by Cx should be much more reduced than the PSs requiring Cy. We feel that the use of comparative saturation effects of photolabeling and phosphoryl transfer using [7-a~P]8-NaATP (Fig. 4), cell fractionation and protein purification procedures separating C~s from PS~s (Fig. 2), and comparison of efficiencies of photoincorporation and photoinhibition of phosphoryl transfer related to specific C~s and PS~s will prove to be extremely useful for resolving biological pathways (e.g., kinase cascades) that are regulated by phosphorylation. In summary, the azido photoprobes allow us to investigate a wide variety of cellular processes on a molecular level. They can assist in the determination of structural characteristics of proteins, regulatory pathways, and cellular location and distribution of specific proteins. As these probes become more widely used, the applications outlined here will certainly be expanded, as will our knowledge of cellular functions regulated by protein-nucleotide interactions.

[53] L a b e l i n g A c y l - C o A B i n d i n g S i t e s w i t h Photolabile Analogs B y ROLAND E. BARDEN, FIDELIS M. ACHENJANG, and

CHRISTOPHER M. ADAMS Principle A general discussion of the rationale and methodology for labeling biological receptor sites with photolabile reagents has been presented. 1 We have prepared photolabile analogs of acyl-CoA by incorporating an arylazido group into the acyl moiety. This approach was followed in the synthesis of p-azidobenzoyl-CoA (PAB-CoA) and S-[12-N-(4-azido2-nitrophenyl)aminododecanoyl]-CoA (AND-CoA). PAB-CoA has been used successfully in photoaffinity labeling experiments with acylCoA:glycine N-acyltransferase (EC 2.3.1.13, glycine acyltransferase) from beef liver) citrate synthase (EC 4.1.3.7) from pig heart, z transcarboxylase [methylmalonyl-CoA:pyruvate carboxytransferase (EC 1 H. Bayley and J. R. Knowles, this series, Vol. 46 [8]. E. P. Lau, B. E. Haley, and R. E. Barden, Biochemistr3' 16, 2581 (1977). 3 E. P. Lau and R. E. Barden, unpublished data, 1977. METHODS IN ENZYMOLOGY, VOL. 91

Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181991J,

634

ACTIVE-SITE LABELING

%

.

:

©N3

[53]

o. o_ ~ _ ~

PAB-coA

H

H 02

: °CH2-~-(CH2~f.-N-.~3 O

NAT-CoA

FIG. 1. Structures of photolabile analogs of acyl-CoA.

2.1.3.1)] from Propionibacterium shermanff, 4 and pyruvate carboxylase (EC 6.4.1.1) from yeastS'6; and AND-CoA has been used in studies with citrate synthase from pig heart 7 and carnithae acetyltransferase (EC 2.3.1.7) from pigeon breast muscle. 8 In some experiments the thiol ester bond in AND-CoA was found to be labile, which led to a loss of radioactivity from labeled protein, since the radiolabeled reagent carries the radioactive element (3H or asS) in the CoA moiety. Consequently, a thioether analog of long-chain acyl-CoA, S-[13-N-(4-azido-2-nitrophenyl)amino-2-oxotridecyl]-CoA (NAT-CoA), has also been synthesized, The structures of the three analogs are shown in Fig. 1. The synthesis of photolabile analogs of a coenzyme is especially attractive, since such reagents can, in principle, be used to investigate the receptor site(s) on each enzyme for which the particular coenzyme is a substrate or effector. According to Mosbach, 9 there are 78 different enzymes that accept CoA as a substrate. Additionally, long-chain acyl-CoA (palmityl-CoA) is known to inhibit a large number of enzymes, at least some examples of which are believed to represent a regulatory process of physiological significance.1°'11 Thus the photolabile reagents described 4 E. M. Poto, H. G. Wood, R. E. Barden, and E. P. Lau, J. Biol. Chem. 253, 2979 (1978). s D. E. Myers and M. F. Utter, Anal. Biochem. 112, 23 (1981). 6 D. E. Myers and M. F. Utter, Fed. Proc., Fed. Am. Soc. Exp. Biol. 37, 856 (1978). 7 C. M. Adams and R. E. Barden, unpublished data, 1978. a F. M. Achenjang and R. E. Barden, unpublished data, 1981. K. Mosbach, Adv. Enzymol. Relat. Areas Mol. Biol. 46, 205 (1978). 10 H. Ogiwara, T. Tanabe, J. Nikawa, and S. Numa, Eur. J. Biochem. 89, 33 (1978). 11 A. V. Caggiano and G. L. Powell, J. Biol. Chem. 254, 2800 (1979).

[53]

LABELING ACYL-COA BINDING SITES

635

here are potentially suitable for labeling studies with a large number of enzymes. Syntheses In our chemical syntheses, water is twice-distilled and other solvents are freshly purified by literature methods. TM In the synthesis of PAB-CoA, 2 p-aminobenzoic acid (Aldrich) is converted to p-azidobenzoic acid via the diazonium salt. la p-Aminobenzoic acid, 1.37 g, is suspended in 50 ml of 4N H2SO4 at 0° and treated with 0.83 g of NaNO2 (1.2 molar excess) dissolved in 12 ml of water. After 10 min, 0.78 g of NaN3 in 12 ml of water is added, followed by stirring for 60 min. The precipitate is collected by suction filtration, washed with ice-cold water, and air-dried (in the dark). Yields of about 80% (8 mmol) are obtained; mp 180-181 °. The esterification of CoA requires an activated form of the acid, a need that is met by synthesis of the N-hydroxysuccinimide ester ofp-azidobenzoic acid. 14N-Hydroxysuccinimide (Aldrich, 0.58 g) and p-azidobenzoic acid (0.81 g) are dissolved in 45 ml of ethyl acetate, to which is added 1.0 g of dicyclohexylcarbodiimide (Aldrich) in 15 ml of ethyl acetate. The solution is left overnight at room temperature with stirring. A white precipitate is removed by filtration, and the filtrate is concentrated by rotary evaporation. Fifty milliliters of hot methanol are added to the concentrate followed in a few minutes by filtration; the filtrate is again concentrated by rotary evaporation. Upon cooling the concentrate, a precipitate forms that is subsequently collected and air-dried (in the dark). Yields of at least 80% ( - 4 mmol) are obtained; m p - 168° (with decomposition). PAB-CoA is prepared by esterification of CoA with a small (~20%) molar excess of the ester. 1~ Thirteen milligrams of CoA (Li ÷ salt, P-L Biochemicals) are dissolved in 1.0 ml of water, to which is added 0.1 ml of 1 M KHCO8 followed quickly by 1.0 ml of acetone containing 5 mg of the N-hydroxysuccinimide ester. More acetone is added in small amounts if needed to maintain a homogeneous solution. The disappearance of thiol groups (CoA) is monitored with 5,5'-dithiobis(2-nitrobenzoic acid). Upon completion of the reaction (-30 rain), acetone is removed with a stream of N2 gas and the aqueous solution is acidified to pH 3-4 with 1 M HCI. The sample is then chromatographed on a Cellex-D column (Bio-Rad; 2.0 × 23 cm) equilibrated with 3 mM HC1; elution is achieved with a 1.5-liter linear gradient of 0.08 12 j. A. Riddick and W. B. Unger, "Organic Solvents." Wiley (Interscience), New York, 1970. 1~ S. H. Merrill and C. C. Unruh, J. Appl. Polymer Sci. 7, 273 (1963). ~4 y. Lapidot, S. Rappoport, and Y. Wolman, J. Lipid Res. 8, 142 (1967). is A. Al-Arif and M. Blecher, J. Lipid Res. 10, 344 (1969).

636

ACTIVE-SITELABELING

[53]

to 0.3 M LiC1 in 3 mM HCI. The PAB-CoA peak ( - 0 . 2 M LiCI) is collected and lyophilized; salt is removed by filtration on a Sephadex G-15 column (2 x 45 cm) in 3 mM HCI. The purity of PAB-CoA is assessed by thin-layer chromatography (TLC) at room temperature on cellulose sheets (Eastman Kodak, No. 13254). With a solvent system of 1-butanol-glacial acetic acid-water (5 : 2 : 3, v/v/v) PAB-CoA has an Re -0.59, and the Rs of CoA and the N-hydroxysuccinimide ester ofp-azidobenzoic acid is -0.42 and -0.95, respectively. The extinction coefficient for PAB-CoA at 265 nm is 22.2 ___ 0.4 mM -1, and absorption spectra at neutral pH, before and after photolysis, have been published. 2 PAB-CoA solutions are stored frozen at -20°; ordinary laboratory glassware is opaque to light of wavelength less than -340 nm; thus, extra precautions to prevent photodecomposition of the reagent are not usually required for solutions in glass containers. The synthetic procedure described above can also be used to prepare p-azidophenylacetic acid3,5 and the o- and m-isomers of azidobenzoic acid. 5 Radioactive PAB-CoA can be prepared by scaling down the chemical synthesis and introducing 14C withp-amino[carboxy1-14 C]benzoic acid (40 Ci/mol, ICN) or 3H with [(G)-aH]CoA (500 Ci/mol, New England Nuclear). Myers and Utter have published an enzymic method for esterifying CoA withp-azidobenzoic acid, using a medium-chain fatty acid: CoA ligase (AMP-forming) (EC 6.2.1.2) from beef liver mitochondria. ~ This method appears to be particularly attractive for the synthesis of PAB-CoA with a very high specific radioactivity. AND-CoA is synthesized by esterification of CoA with the N-hydroxysuccinimide ester of 12-N-(4-azido-2-nitrophenyl)aminododecanoic acid. The arylazido acid is prepared by a procedure based on that described by Guillory and Jeng. TM Ten milliliters of water and 4 ml of ethanol are mixed with 335 mg of Na2 CO3 and 226 mg of 12-aminododecanoic acid (Aldrich) in a round-bottom flask. After gentle heating to dissolve the components, 130 mg of 4-fluoro-3-nitrophenyl azide (Pierce) is added in 5 ml of warm ethanol. The solution (pH -10) is heated at 50-52 ° in an oil bath for 25-30 hr with stirring; a cooling condenser is attached. The solution is concentrated by removing ethanol with a rotary vacuum evaporator and then diluted with 15 ml of water prior to extraction twice with 80 ml of diethyl ether. The water layer is acidified to pH 3.0 with 3 M HC1, and the product is then extracted with two 80-ml batches of diethyl ether. The ether extracts are combined, washed with water saturated with NaC1, dried with anhydrous Na2SO4, and evaporated to ~8R. J. Guilloryand S. J. Jeng, this series, Vol. 46 125].

[$3]

LABELING ACYL-CoA BINDING SITES

637

yield the product. The emulsions sometimes encountered in extraction steps are broken by centrifugation in a desk-top centrifuge. The yield is - 7 0 % (0.5 mmol) based on 4-fluoro-3-nitrophenyl azide; mp 94-96°; characteristic NMR, UV-VIS, and IR spectral data have been reported. TM The N-hydroxysuccinimide ester of 12-N-(4-azido-2-nitrophenyl), aminododecanoic acid is prepared by dissolving 70 mg of the acid and 44 mg of N-hydroxysuccinimide in 25 ml of ethyl acetate, to which is added 80 mg of dicyclohexylcarbodiimide. The system is left for 3 hr at room temperature with stirring. The white precipitate is removed by suction filtration, and the filtrate is carefully evaporated to dryness under vacuum. The bright orange residue is crystallized from 95% ethanol; yield is - 6 5 % (0.14 mmol); mp 46-48°; mass spectrum, m/e 474. The synthesis, handling, and storage of long-chain CoA ligands are performed in glassware silanized with dimethyldichlorosilane. In the preparation of AND-CoA, CoA-Li÷ salt (23 mg) and the Nhydroxysuccinimide ester (36 mg, 2- to 3-fold molar excess) are added to 3 ml of 0.1 M KHCOz-acetone (2 : 5, v/v) and stirred for 3 hr at room temperature. Acid-insoluble material is precipitated with ice-cold 5% HC104, collected by centrifugation, and washed with small volumes of 0.8% HC104, absolute ethanol-anhydrous diethyl ether (1 : 9, v/v), and finally anhydrous diethyl ether. The pellet is dissolved in 50 mM K ÷ phosphate, pH 6.8, and stored frozen. Yields are 30-40%'based on CoA; = 42.6 mM -1 at 259 nm. To initiate the synthesis of NAT-CoA, 12-N-(4-azido-2-nitrophenyl) aminododecanoic acid is converted to the acid chloride by dissolving 370 mg of the acid in 2 ml of chloroform and adding 0.1 ml of thionyl chloride (freshly distilled) and 0.1 ml of dimethylformamide as catalyst. The reaction mixture is vented through a CaC12 drying tube and left at room temperature for a day. Occasional flushing of the system with N2 removes evolving gases; an additional 0.1 ml of thionyl chloride is added after a half day of reaction time. The solution is concentrated by rotary vacuum evaporation (using drying tubes to prevent contamination with moisture) and then washed with small volumes of petroleum ether (bp 30-60°). The residue is dissolved in 2 ml of chloroform at 4° and treated immediately with freshly generated ethereal diazomethane. T M After addition of the diazomethane, the system is allowed to warm to room temperature and left for several hours. Solvent is removed by vacuum distillation. The residue is a mixture of 13-N-(4-azido-2-nitrophenyl)amino-2-oxotridecyl ~TThe preparation of diazomethane and HAZARDS associated with its use have been described: F. Arndt, "Organic Syntheses" (A. H. Blatt, ed.), Vol. II, p. 165. Wiley, New York (1943). Also: M. T. Bush and E. Sanders-Bush, Anal. Biochem. 106, 351 (1980). is S. S. Husain and G. Lowe, Biochem. J. 108, 855 (1968).

638

ACTIVE-SITE LABELING

[53]

chloride and the analogous diazomethyl ketone compound. (The methyl ester of the parent acid is sometimes present, also.) 1H NMR (CDCI3): 3.66 (s, 1H, COCHN2), 4.08 (s, 2H, COCH2C1); 13C NMR (CDCLz); 51.32 (s, --CHN2), 36.86 (s, --CHsC1). The thioether analog of long-chain acyl-CoA (i.e., NAT-CoA) is prepared by treating CoA with the mixture of diazomethyl ketone and chloromethyl ketone compounds, following the procedure described for AND-CoA. Yields are - 3 0 % , ~ = 42.6 mM -~ at 259 nm. Purity of AND-CoA and NAT-CoA is assessed by TLC, as described for PAB-CoA. AND-CoA and NAT-CoA each have an Rs of 0.70, whereas the diazomethyl ketone and chloromethyl ketone starting materials migrate at the solvent front; all compounds containing the 4-azido2-nitrophenyl moiety are orange in color. Absorption spectra of NAT-CoA at neutral pH, before and after photolysis, are essentially the same as spectra reported for "arylazido-/3-alanine ATP. ''16 Absorption spectra for AND-CoA, before and after photolysis, are shown in Fig. 2.

0.8

0.6 W C) Z

O

O3 m ,<

0.4

0.2

240

I I 260 280 WAVELENGTH, nrn

300

FIG. 2. Absorption spectra of AND-CoA before and after photolysis. A N D - C o A , 21 Ix M, in 50 m M potassium phosphate, 1 m M EDTA, pH 7.5, was irradiated in a quartz cuvette with a Mineralight UVS-I1 lamp from a distance of - 5 cm for the time indicated on the curves (in minutes).

[53]

LABELING ACYL-CoA BINDING SITES

639

Radioactive AND-CoA and NAT-CoA have been synthesized from [aH(G)]CoA or [asS]CoA, using reduced quantities and volumes in the above procedure. Since the reaction volume is small, the product is isolated by TLC. The solution is spotted on cellulose plates (without fluorescent indicator; EM Reagents, No. 5537) and carefully dried with a stream of warm air. The plate is developed in the solvent described above; after drying, the orange material ofR~ - 0.7 is scraped into a 15-ml centrifuge tube. The product is extracted from the cellulose with 3 ml of methanol acidified with 3 or 4 drops of 2 M HC1; after stirring for several minutes, the cellulose is sedimented by centrifugation. The supernatant is removed and concentrated to 1 ml, after which the product is precipitated with 9 volumes of diethyl ether. [asS]CoA is synthesized enzymically by dried cells of B r e v i b a c t e r i u m a m m o n i a g e n e s as suggested by Shimuzu et a1.,1° but generally following the conditions for small-scale preparations as described by Hosoki et al. 20 The reaction mixture, pH 6.57, contains in 15 ml: 400/xmol of ATP, 96/~mol of L-[asS]cysteine • HC1 (Amersham) (specific activity ~ 18 Ci/mol), 100/.,mol of MgSO4, 100/zmol of sodium pantothenate, 1.5 mmol of potassium phosphate, 20 mg of sodium laurylbenzene sulfonate, and 1 g of dried cells. After incubation at 37-40 ° for 15 hr with shaking, CoA is isolated as described by Hosoki et al. z° The yield is - 11 mg of [~S]CoA (specific activity --- 9000 cpm/nmol). Photoaffmity Labeling A volume of this series was devoted to affinity labeling and contains a chapter on the general methodology of photoaffinity labeling as well as several on specific applications. 21 An example of a prime target for investigation of acyl-CoA binding sites by photoaffinity labeling with PAB-CoA and AND-CoA or NAT-CoA is citrate synthase from pig heart. This enzyme uses acetyl-CoA as a substrate (potential for active site labeling) and is inhibited by palmitylCoA via an apparently site-specific allosteric process (potential for regulator site labeling). In addition, the amino acid sequence of citrate synthase has been reported by Bloxham et al. zz The availability of the amino acid sequence is a notable convenience in site-labeling studies, since isolation of the labeled (tryptic) peptide(s) followed by amino acid analysis should suffice to identify the labeled residue(s). Preliminary experiments we have performed with citrate synthase will be described as a 19 S. Shimuzu, Y. Tani, and K. 0gata, this series, Vol. 62 [44]. 20 K. Hosoki, S. Kurooka, and Y. Yoshimura, Radiodisotopes 21,502 (1972). 21 This series, Vol. 46. 22 D. P. Bloxham, D. C. Parmelee, S. Kumar, R. D. Wade, L. H. Ericsson, H. Neurath, K. A. Walsh, and K. Titani, Proc. Natl. Acad. Sci. U.S.A. 78, 5381 (1981).

640

ACTIVE-SITE LABELING

[53]

TABLE I PHOTOAFFINITY LABELING OF CITRATE SYNTHASE WITH PAB-CoA a-b

(a) (b)

PAB-CoA 0zm)

OAA (raM)

CoA (~M)

% Activity remaining

400 400 80 80 80

0 1.0 1.0 1.0 1.0

0 0 0 100 500

86 67 75 84 98

The incubated system contained 21 ~g of citrate synthase in 0.15 ml of 30 mM potassium phosphate, pH 7.3, with the concentrations of PAB-CoA, oxaloacetate, and CoA as listed above. The sample was held in the well of a spot plate and irradiated for 5 min at 4° with a Mineralight UVL-21 lamp from a distance of - 2 cm. Ten-microliter aliquots were removed and diluted to 0.25 ml with 50 mM HEPES, 0.5 mM EDTA, 0.1 M NaCI, pH 7.8. Ten microliters of the dilute sample were assayed for activity as described by P. A. Srere in this series, Vol. 13 I1]. OAA, oxaloacetate. b From Lau and Barden. z

means of illustrating the application of photolabile acyl-CoA analogs to enzymes a,r An important first step in active-site labeling is to demonstrate that the reagent (PAB-CoA) is active-site-directed. With citrate synthase we previously showed that benzoyl-CoA (a close structural analog of PABCoA) is a linear competitive inhibitor versus acetyl-CoA with a Ki of 21 /zM. 2a A second important factor is that reaction conditions for pbotolabeling must be designed to enhance binding of the analog. Thus, while binding of substrates to citrate synthase is basically random, acetyl-CoA binds to the enzyme • OAA complex with 20 times greater affinity than to the free enzyme. 24 In (a) of Table I, one notes that a saturating level of PAB-CoA produces a significant increase in photoinhibition when oxaloacetate is present. A third consideration is that a ligand that binds reversibly at the acyl-CoA portion of the active site should protect the enzyme against photoaffinity labeling. This point is illustrated in (b) of Table I, where the presence of added CoA leads to a protection against observed inhibition. z3 M . S . Owens and R. E. Barden, Arch. Biochem. Biophys. 187, 299 (1978). 24 G. Peterson, Eur. J. Bochem. 46, 1 (1974).

[53]

LABELING ACYL-CoA BINDING SITES

641

TABLE I! IRREVERSIBLE INHIBITION OF CITRATE SYNTHASE WITH AND-CoA a

Site protectors None

OAA

Acetyl-CoA

Citrate

AND-CoA (l,zM)

-hv

+hv

-hv

+hv

-hv

+hv

-hv

+hv

0 1.7 3.4 8.5

100b 90 84 74

100 84 73 38

100 100 100 100

100 100 100 100

100 91 84 76

100 79 73 48

100 96 86 76

100 86 74 46

a The incubated system contained 8 #g of enzyme in 0.5 ml of 40 mM potassium phosphate, 1 mM EDTA, pH 7.5, with the concentrations of AND-CoA listed. When present, the concentrations of oxaloacetate, acetyl-CoA, and citrate were 97 tzM, 48 txM, and 2 mM, respectively. Samples were incubated in a quartz cell at 4° for 4 rain prior to assay of activity (see Table I). When phototyzed (+hv), samples were irradiated for 3 min in a locally constructed photoreactor consisting of 16 lamps (GE G8T5) equally spaced on the perimeter of a cylinder 18 cm in diameter. b Percentage of activity remaining.

As is the case with labeling active sites, an important step in regulator-site labeling is to demonstrate that the photolabile analog (AND-CoA or NAT-CoA) is regulator-site-directed. With citrate synthase we have shown that AND-CoA closely mimics palmityl-CoA as an inhibitor of catalytic activity. Concentrations of AND-CoA in the low micromolar range rapidly and irreversibly inhibit the enzyme in the absence of photolysis; oxaloacetate provides complete protection against the ligand-induced inhibitory process, whereas acetyl-CoA or citrate provide little or no protection (Table II). These observations are the same as reported for palmityl-CoA, n,25 When the system is photolyzed, the inhibition produced by AND-CoA increases; oxaloacetate again provides complete protection with little or no protection from acetyl-CoA or citrate (Table II). These preliminary results indicate that AND-CoA (and thus palmityl-CoA) cannot bind to the enzyme • OAA complex, since no inhibition is observed upon photolysis in the presence of oxaloacetate. Thus oxaloacetate protects by preventing the binding of the regulator (longchain acyl-CoA) rather than by stabilizing an enzyme • regulator complex. Also, the preliminary results show that the primary inhibitory process, even in photolyzed systems, is a ligand-induced irreversible conformational or structural change. Photoincorporation of the analog enhances the ~s p. A. Srere, Biochim. Biophys. Acta 106, 445 (1965).

642

ACTIVE-SITE LABELING

[53]

inhibition b y a significant amount; nonetheless, the o b s e r v e d inhibition upon photolysis is the result of two distinct inhibitory processes. Covalent bond formation b e t w e e n the photoactivated reagent and the e n z y m e is often not an efficient process. The data in (a) of Table I show that citrate synthase is only - 33% inhibited after photolysis in the presence of a saturating level of PAB-CoA. A similar result was obtained when an acyltransferase was photolabeled with PAB-CoA2; h o w e v e r , photoincorporation of PAB-CoA into t r a n s c a r b o x y l a s e was quite efficient. 4 The specificity with which PAB-CoA labels an active site residue(s) on citrate synthase has yet to be determined. In the literature, only one report has described specific photolabeling of a single residue in a binding site. 26 Generally, labeling of multiple residues can be anticipated. Acknowledgment This chapter is based on work supported by National Institute of Health Grant GM 20077.

26 A. R. Kerlavage and S. S. Taylor, J.

Biol. Chem.

255, 8483 (1980).