8-Azido-2'-O-dansyl-ATP - Journal of Biological Chemistry

THEJOURNALOF BIOLOGICAL CHEMISTRY Q 1989 by The American Socxety for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 14, Issue of May 15, pp. 7981-7988.1989 Printed in U.S.A.

8-Azido-2'-O-dansyl-ATP AFLUORESCENTPHOTOAFFINITYREAGENT APPLICATION TO ADENYLATE KINASE*

FOR ATP-BINDINGPROTEINS

AND ITS

(Received for publication, June 6, 1988)

Hua Chuan, Johnson Lin, and Jui H. WangS From the Bioenergetics Laboratory, Acheson Hall, State University of New York, Buffalo, New York 14214-3094

The photoaffinity reagent8-azid0-2'-0-['~C]dansyl- kinases could be covalently labeled with reactive analogues of ATP (AD-ATP) has been synthesized for labeling and ATP and ADP (1-7, 12). In practice, it is desirable to intromonitoring the active sitesof ATPases and kinases. In duce only minor changes in the structure of ATP or ADP in its first application, the reagent isused to explore the designing the analogues so that theanalogue will bind to the active site of adenylate kinase from rabbit muscle. In active site almost as well as the natural substrate or effector the dark,AD-ATP inhibits adenylate kinase reversibly and that thelabeling reaction will have high specificity. and competitively with KI= 0.25 f 0.01 NM. Under The most widely used reactive analogues of adenine nucleweak UV illumination, AD-ATP labels adenylate ki- otides are azido-ATP and azido-ADP which rapidly change naseirreversibly.The photoinactivation data also under weak ultraviolet light to the corresponding nitrenes show KI = 0.25 % 0.02 pM. that can react with all amino acid residues. Because of their The ratio ( r ) of the specific activity of AD-ATP- anti-conformation, 2-azido-ATP and 2-azido-ADP were often labeled adenylate kinase to that of the unlabeled enzyme has been determined as a function of the number chosen (9) for labeling the active site of FI-ATPAse in pref(n) of label/enzyme. The linearplot of F uersus n with erence to 8-azido-ATP and 8-azido-ADP which have synslope equal to -1 shows that the labeling is very spe- conformation (10). In thepresent work, the fluorescent photo(AD-ATP)' cific, i.e. each label completely inactivates an enzyme affinity reagent 8-azid0"2'-0-1'~C]dansyl-ATP molecule. After the labeled enzyme was partially hy- has been synthesized and its application to adenylate kinase drolyzed and the radioactive peptides analyzed and is reported. sequenced, it was found that Leu-115, Cys-25, and EXPERIMENTALPROCEDURES probably His-36 were labeled, in agreement with previous conclusions on the structureof the active site of Materials this enzyme based on amino acid sequence, x-ray difADP (sodium salt), AMP, ATP (disodium salt), 8-azido-ATP (sofraction, andNMR studies. The environment-sensitivefluorescent dansyl group dium salt), NADH, P-enolpyruvate, pyruvate kinase, lactic dehydroof AD-ATP can function as an in situ probe for moni- genase, adenylate kinase from rabbit muscle, pepsin, Hepes, and dansyl chloride were purchased from Sigma. ["CIDansyl chloride of toring ligand or conformation changes at the active specific activity 111 mCi/mmol was supplied by Research Products site. The fluorescence of AD-ATP-labeledenzyme with International Corporation. n = 0.9 is not affected by ATP but increases with the concentration of AMP in solution. This observation is Methods also in agreement with the previous conclusion that Synthesis of 8-Azido-2'-O-dansyl-ATP and 8-Azid0-2'-0-['~C] ATP does not bind to theAMP site of adenylate kinase. dansyl-ATP (AD-ATP)"In a typical experiment, 6.2 mg of 8-azidoThe observed enhancement of fluorescence indicates ATP was dissolved in the dark in 1.67 M NazC03/NaHC03 buffer at that binding of AMP by this enzyme causes environ- pH 9.8 and 0 'C to form a clear solution containing 30-40 mM 8azido-ATP. A supersaturated solution of 2 mg of dansyl chloride in mental change at itsATP site. The possible usefulness of AD-ATP as an effective 0.5 ml of acetone at 0 "C was mixed with the above solution in the biological inhibitor or as a molecular probe for study- dark with stirring. The molar ratio of 8-azido-ATP to dansyl chloride ing the structureand regulation of ATP-binding pro- was 1:10 for nonradioactive dansyl chloride or k1.2 for ['4C]dansyl chloride. The reaction mixture was kept at 0-4 "C for 1 h and then teins is discussed.

Analogues of ATP and ADP with reactive functional groups have become increasingly useful because of the growing number of vital enzymes that need ATP as substrate or as an effector for metabolic control. In principle, all ATPases and

at 28 "C for 10-18 h until dansyl chloride was no longer detectable in the mixture by TLC analysis. The cloudy mixture was then separated on TLC (Baker Si-250) with the solvent mixture 1-butanol/H20/ CH3COOH (10:6:3). The AD-ATP band ( R F = 0.38) was scraped off and extracted with H20/ethanol (10:3). The extract was lyophilized and assayed. The molar ratio of the dansyl group to phosphate in 8azido-2'-O-dansyl-ATP as determined by dansyl absorbance at 339 nm and phosphate analysis was found to be 1:3.0. The molar ratio of the dansyl group to the adenine group in dansyl-ATP prepared by the same procedure was found to be 1:l.O from the observed absorbance at 259 and 339 nm of 0-dansyl-ATP andof the model compound dansyl-OCHsCH3. The 'H NMR spectrum of AD-ATP is shown in Fig. 1. A comparison with the 'H NMR spectra of ATP, 3'-0-(2,4,6-trinitrocyclohex-

* This work wassupported by Research Grant BRSG SO RR 07066 from the Biomedical Research Support Program, Division of Research Resources, and Research Grant GM 41610from the National Institute of General Medical Sciences, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adver' The abbreviations used are: AD-ATP, 8-azido-2'-O-dansyl-ATP; tisement" in accordance with 18U.S.C. Section 1734 solelyto indicate Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; OPA, othis fact. phthalaldehyde; HPLC, high performance liquid chromatography; $ To whom correspondence should be addressed. PTH, phenylthiohydantoin.

7981

7982

A Fluorescent Photoaffinity Reagent

f

FIG. 1. Proton NMR spectrum of 8-azido-2'-O-dansyl-ATP. About 2 mg of TLC-purified AD-ATP was dissolved in DzO and adjusted to pH 6.5-7.0 with a solution of 30% NaOD in DzO. After lyophilization, the dry residue was redissolved in DzO, and the solution was lyophilized again. This procedure was repeated twice to replace all the exchangeable 'H by'H. The final dry residue was dissolved in dimethyl sulfoxide/D20 mixture (1090, v/v) for 'H NMR measurement. All operations were performed in a dark room with dim red light. The NMR mectrometer (Gemini-300) wasDurchased with funds of a matching grant from the National Science Foundation (CHE-8613066). adieny1idene)adenosine and 3'-0-(5-fluoro-2,4-dinitrophenyl)ADP ether indicates that the dansyl group in AD-ATP is attached to the 2'-0 instead of the 3'-0 position of the ATP moiety. For 3'-0-(5fluoro-2,4-dinitrophenyl)ADP,the chemical shift difference between H2, and H3' is A6 = 0.05 ppm which is much smaller than that for ADP ( 6 ~ ~ .6~~ = 0.43). This is presumably due to the electron withdrawing effect of the fluorodinitrophenyl group attached to the 3'-O-position which causes the magnetic resonance of HY to shift downfield (10). Similar observation was reported earlier for 3'-0(2,4,6-trinitrophenyl)adenosine(11).In both cases, the 'H NMR data suggest that thesubstituent groups are attached to the 3'-0position of the ribose. Onthe other hand,the chemical shift difference between H2' andHs. of AD-ATP (&,. - 6 ~ =. 0.32) is larger than thatof ATP (A6 = 0.10 ppm). This is consistent with an electron withdrawing dansyl group attached to the 2-O'-position of ribose whichcauses the magnetic resonance of Hn' toshift downfield. Enzymatic Assays-The specific activity of adenylate kinase was determined at 30 "C by an ATP-regenerating system in 50-60 mM Hepes/NaOH buffer, pH 7.6, containing 1.2 mM ATP, 1.4 mM AMP, 1.0 mM P-enolpyruvate, 1.2 mM MgC12,142 mM KCl, 0.3 mM NADH, 20 units/ml pyruvate kinase, and 11 units/ml lactic dehydrogenase. The steady-state rate of ATP consumption was computed from the observed linear decrease of As40 with time due to thecoupled oxidation of NADH. Protein concentrations were normally determined by the Coomassie Blue binding method (13). Radioactivity was assayed by liquid scintillation counting. The specific activity of control unlabeled adenylate kinase was 431Nmol of ATP min" mg". Photoaffinity Labeling with AD-ATP-Quartz cuvettes containing the reaction mixtures were illuminated by 254/365 nm UV light in a Chromato-vue cabinet which provided radiation intensity of25 microwatts/cm2a t 254 nm. Under weak UV illumination the irreversible

inactivation of adenylate kinase by AD-ATP was followed conveniently by removing aliquots of the reaction mixture at different illumination periods, gel filtration of the samples to remove the free as well as noncovalently bound nucleotides, and assaying the specific activity (A) of each sample. Although the free AD-ATP molecules in the reaction mixture were also decomposed by ultraviolet light, it was found that under the experimental conditions the activity ratio r = A/Ao, where Ao represents the specific activity of the unlabeled enzyme, decreased with time precisely according to the pseudo-first order equation -d In r/dt = k' or In r = -k't. Experimental values of this photoinactivation rate constant k' at various concentrations of ATP and AD-ATP were determined from the In r versus t plots. The location of the [14C]AD-ATPphotolabel in adenylate kinase was determined by pepsin cleavage of the labeled enzyme, followed by isolation and sequencing of the radioactive peptides as described previously (14). The fluorescence of AD-ATP-labeled adenylate kinase was measured at 470 nm with 330 nmexciting light in a HitachiPerkin-Elmer fluorescence spectrometer (MPF-2A) (15). RESULTS

Reversible Inhibition in the Dark-The rapid photoaffinity labeling of adenylate kinase by AD-ATP is illustrated in Fig. 2. A t zero illumination time, all three mixtures ( A , B,and C ) exhibit about the same kinase activity as themixture kept in the dark ( B I ) or the mixture illuminated in the absence of AD-ATP ( A I ) . This observation shows that the inhibition observed by our experimental procedure was completely due to the photolabeling by AD-ATP, none due to noncovalently bound AD-ATP. In thedark, AD-ATP inhibits theadenylate

A Fluorescent Photoaffinity Reagent 600

7983

I

illumination time (min) FIG.2. Photoinactivation of adenylate kinase by AD-ATP. Each of the following mixtures in buffer A was preincubated for 15 min in the dark at 23 & 1 "C before the photoreaction was started A I , 50 p M adenylate kinase + 1.2 mM ATP; A, 40 pM adenylate kinase + 1.2 mM AMP andthen 68 pM AD-ATP; BI,40 p M adenylate kinase + 1.2 mM ATP and then +68 p~ AD-ATP, in the dark; E , same as E l , but not kept in the dark; C,42 p~ adenylate kinase + 72 p M AD-ATP, The reaction mixtures in polypropylene containers were illuminated by 25 microwatts/cm* 254 nm W light for 3, 5, 10, 20, and 60 min, 1-pl aliquots were taken, and each was mixed with 2 ml of the assay medium containing 1.2 mM ATP. After 3-5 min, 3 p l of 1 M MgC12 solution was added to initiate the assay reaction. The assay was performed as described under "Methods."

kinase reversibly and competitively according to the well known relationship

where [I] and Kl denote the concentration and dissociation constant, respectively, of AD-ATP. The data in Fig. 3A give K, = 0.14 mM and Kr = 0.25 PM. Thus, AD-ATP is bound to the ATP site of adenylate kinase much more tightly than ATP itself. The steric hindrance between its 8-azido and 2'0-dansyl groups could force AD-ATP into the anti-conformation and make it a much better inhibitor than 8-azidoATP. Nuclear Overhauser effect spectra show that AD-ATP in solution is indeed in anti-conformation (data not shown). The hydrophobicity of the dansyl group may be partly responsible for making the KI of AD-ATP 500-fold smaller than the K , of ATP itself. Irreversible Inhibition by Photoaffinity Labeling-ATP competes with AD-ATP for the same binding site inadenylate kinase and protects the enzyme from irreversible inactivation by this photoaffinity reagent. Therefore, ATP acts as a competitive inhibitor of this photoinactivation reaction so that the rate of irreversible photoinactivation reaction is given by

The kinetic data of irreversible photoinactivation of adenylate kinase by AD-ATP summarized in Fig. 3B give K, = 0.14 mM and KI = 0.26hM, in agreement with the values computed from Fig. 3A. For reaction mixtures in polypropylene containers irradiated with 25 microwatts/cm2 254 nm UV light, kkaXwas found to be 0.25 rnin". The intensity of the UV light was deliberately kept low so that the labeling reaction was slow enough to be monitored conveniently. Specificity of Labeling-Thedegree of inhibition can be correlated with the number of covalent labels attached to adenylate kinase by using 8-azido-2'-0- ['4C]dansyl-ATP.The

20

0

l/[ATPl

40

(mM")

0

4

1/ [AD-ATP]

FIG.3. Competitive inhibition of adenylate kinase by ADATP. A, reversible inhibition in the dark. The assay mixture contained 19.1 nM adenylate kinase, 0.02-125 mM ATP asindicated, and 0,0.5,2, or 5 p~ AD-ATP. After incubation for 2-3 min at 30 "C, the reaction was started by addition of 3 pl of 1.0 M MgCl, (aqueous) to 2 ml of the incubated mixture. A11 manipulations were performed in B, the dark. [AD-ATP] = 0 (Ut, 0.5 p~ (A), 2 p~ (O), and 5 p~ (8). irreversible inhibition under weak UV illumination. Adenylate kinase (19.1 nM) was preincubated with 0-15 p~ AD-ADP as indicated and 0,0.25, or 1.0 mM ATP at room temperature (28 f 1 "C) in the dark for 15 min. After illumination with UV light for 1, 2, 4, 8, or 16 min, the samples were taken out and assayed for specific activity. As discussed under "Methods," the pseudo-first order rate constant for the labeling reaction was determined as -d In r/dt, where r = ratio of the specific activity of the labeled adenylate kinase to that of the unlabeled enzyme. [ATP] = 0 (O), 0.25 (0),and 1.0 (0)mM. data presented in Fig. 4A show that the affinity labeling is highly specific,i.e. every AD-ATP label is covalently attached to the active site. The reactive nitrenecan be covalently attached to any one of the amino acid residues of the active site and completely inactivates the enzymemolecule. The efficiency of nitrene insertion depends on the nature and location of each residue. The radioactive label also allows us to identify the protein functional groups present at the active site. By partial hydrolysis of the labeled enzyme with pepsin, separation of the radioactive peptides as illustrated in Fig. 4B, and sequencing each isolated radioactive peptide, it is possible to identify the labeled amino acid residues by comparing the sequences of the radioactive peptides with the complete sequence of rabbit muscle adenylate kinase (16). Identification of ['4C]AD-ATP-labeled Region and Amino Acid Residues-Fig. 4B shows that four major radioactive fragments were isolated from the pepsin digest of [I4C]ADATP-labeled adenylate kinase (n = 0.95) by means of HPLC on a pair of CIS columns connected in tandem. These four fragments contained 73% of the total radioactivity of the labeled enzyme. Specifically, 15% was associated with peptide P1,24% with peptide P2,14% with peptide P3, and 20% with peptide P4. Aliquots of each peptide were completely hydrolyzed in 6 N HCl, the hydrolysates were treated with OPA, and the OPA derivatives were separated on a C18 column and identified. The results of amino acid analyses are summarized in Table 1. P1 was composed of Thr and Leu (molar ratio 1:l.g); P2 contained Glu, Lys, Ile, and Val (1:l:l:l); P3 contained Val,Lys, Tyr, Thr, Gly, and His (1:1:2:1:1:1); P4 contained Glu, Lys, Ile, Val, Tyr, Thr, Gly, and His (1:2:1:1:21:1:1). When the complete amino acid sequence of adenylate kinase (16) was taken into account, it seems that

A Fluorescent Photoaffinity Reagent

7984

TABLEI Amino acid compositionof ['4C]AD-ATP-labeled peptides PI, P2, P3, and P4from ['4C]AD-ATP-lnbeled adenylnte kinase from rabbit muscle The molar ratios listed are relative to Val. The expected molar ratios are based on the known amino acid sequence of adenylate kinase, i.e. Pro-Thr-Leu-Leu-Leu for P1 and Cys-Glu-Lys-Ile-ValHis-Lys-Tyr-Gly-Tyr-Thr-His for P4 (16). P1 P2 P3 P4 Amino ExExEXacid Found ExDected Found pected Found pected Found Dected

MINUTES FIG. 4. Specific photoaffinity labeling of adenylate kinase by ['*C]AD-ATP. A, linear correlation between the inhibition of adenylate kinase activity and thenumber of AD-ATP-label/enzyme. r = ratio of the specific activity of the AD-ATP-labeled adenylate kinase to that of the unlabeled enzyme; n = moles of the covalent label/mole of the enzyme. A 3.3 p M adenylate kinase solution in buffer A was preincubated with 19.3 p~ [14C]AD-ATP(42.6mCi/ mmol) at 28 & 1 "C for 15 min in the dark. A control solution in buffer A without [%]AD-ATP was preincubated similarly. After 0, 3, 6, 8, 12, 16, or 20 min of illumination by UV light, a 2 4 aliquot was removed from the irradiated solution, mixed with 2 ml of assay mixture which contained 1.2 mM ATP but no M P , and incubated for 3-5 min to free the noncovalently bound AD-ATP by exchange with medium ATP. The assay was then initiated by the injection of 6 +I of 1 M MgCl2 (aqueous). For the determination of n, a 25-pI aliquot of the reaction mixture was mixed with an equal volume of 0.5% bovine serum albumin solution in buffer Aand gel filtered through Sephadex G-25-80. The filtrate was assayed for both radioactivity and enzyme activity. The concentration of adenylate kinase was obtained by multiplying 3.3 pM by a dilution factor which was determined as theratio of the enzyme activity in 2 liters of the filtrate to that in 2 liters of the solution before gel filtration. B, separation of major radioactive peptides from [14C]AD-ATP-labeledadenylate kinase. About 36 nmol of the labeled adenylate kinase with n = 0.95 was dialyzed against 1%formic acid at 4 "C overnight. The dialyzed sample was digested with 10 ng of pepsin at 30 "C for 5 h. The digested sample was lyophilized and redissolved in 300 pl of 0.1% trifluoroacetic acid. A 15 *I-aliquot of the solution was applied to a double CIS Nova-Pak column which had been preequilibrated with Solvent A, and eluted with a linear gradient as illustrated at a flow rate of 1.0 ml min-I. Fractions were collected manually as indicated by the absorption peaks at 220 nm and only non-overlapping peaks were collected. One-tenth of each fraction was assayed for radioactivity. Solvent A: 30% acetonitrile, 0.1% morpholine, 0.125% trifluoroacetic acid solvent B: 80% acetonitrile, 0.1% morpholine, 0.125% trifluoroacetic acid.

1.0" Pro 1 1.01 1 1.06 1 0.92 1 Thr 1.9 3 Leu -0.4' 1 1 0.2" CYS Glu 1.01 1 1.01 1 1.09 1 0.93 1 1.80 2 LYs Ile 1 0.97 1 0.98 Val 1.01 1 1.0 1 1.0 1 His 1.27 2 1.19 2 2 1.82 2 2.03 TYr 1.06 1 0.92 1 Glv "Assayed by A254 of the PTH-derivative from the 1st Edman degradation cycle of S-carboxymethylated P2. About 1nmol of lyophilized P2 was dissolvedin 20 r l of 1.0 M NaHC03/Na2C03buffer, pH 8.5, and incubated with 1~1 of 10 mM iodoacetate at 40 "C for 2 h in the dark. After quenching by a small amount of 0-mercaptoethanol, the mixture was lyophilizedand thensubjected to Edman degradation in the usual way. 'Assayed byA254of the PTH-derivative from the 1st Edman degradation cycle of P4.

react with OPA, 2) when any amino acid is labeled, the characteristic retention time of its derivative on the HPLC column is altered. The above tentative assignments were supported by direct sequencing of P1 (Fig. 5) and P4(Fig. 6). Edman degradation of P1 proceeded for 5 consecutive cycles and revealed the sequence Pro-Thr-Leu-Leu-Leu. Except for the Pro, which was detected as its PTH derivative by absorbance at 254 nm, the other residues were detected as OPA derivatives because of the greater sensitivity of fluorescence assay. Notably, the 4th cycle was unusual in that only a very small peak of Leu and a smear of undefined peak with longer retention time were detected. The most probable interpretation of this observation is that much of Leu-115 was labeled by the insertion of nitrene generated from the bound AD-ATP with UV irradiation. The detection of radioactivity in the organic phase of the 4th cycle which extracted PTH-Leu also supports this interpretation (Table 11).The possibility of a labeled Tyr-117 following Leu-116 was excluded, because noradioactivity was found in the aqueous phase of the 5th cycle. Edman degradation of P4 was carried out to the 8th cycle. No characteristic amino acid peak was detected in the 1st cycle, but subsequent cycles identified the amino acid sequence Glu-Lys-Ile-ValHis-Lys-Tyr. By comparison of this sequence with the complete amino acid sequence of adenylate kinase, it can be deduced that the 1st amino acid residue of P4 must be Cys25. When P2, which wasequivalent to theN-terminal segment of P4, was treated with iodoacetate before being cleaved by phenylisothiocyanate and trifluoroacetic acid, there was a small peak of S-carboxymethylated Cys and some unidentified P2 and P3 are probably further cleaved products from the peaks of shorter retention time. Moreover, radioactivity was longer peptide P4 which spanned from Glu-26 to Thr-35, detected in the organic phase of the first cycle. Therefore, we whereas P1 probably contained Thr-113 to Leu-116. However, may conclude that Cys-25 was labeled by [14C]AD-ATP.NO P1 might actually be the hexapeptide Pro-112 to Tyr-117 and other labels were found up to the 8th cycle of P4. When the P4 might be the dodedapeptide Cys-25 to His-36 for the remaining portion of P4 washydrolyzed in 6 N HC1, the following reasons: 1) Cys and Pro often preclude detection resulting amino acids were Gly, Tyr, Thr, andHis (1:1:1:0.3). due to the ease of oxidation of Cys and the failure of Pro to The diminished amount of His is unlikely to be due to

7985

A Fluorescent Photoaffinity Reagent

I

Analysis

IT

f

3rd Cycle

FIG. 5. Analysis andsequencing of radioactive peptide P1 from ["C] AD-ATP-labeled adenylate kinase. The separation of OPA derivatives by HPLC was the same as in Fig. 4. The Edmandegradationandderivatization procedures were described previously (14). The productof first Edman degradation cycle was identified as PTH-proline by its elution timet and absorbance at 254 nm. The product of all other cycles were identified by the elution time and fluorescence (f) of the OPA derivatives. For the first Edman cycle, the PTH derivative was dissolved in eluentA, loaded into Cln column, and eluted at 1 ml min" as follows: 0-1 min, 100% eluent A; 15.5 min, 0-40% eluent B; 5.5-12 min, 40% eluent B. Eluent A: 30 mM sodium acetate (pH LO)/acetonitrile (51); eluent B: isopropanol/H*O (3:2). The HPLC control witha blank did not show the small peak due to PTH-proline. Oneletter amino acid code is used.

I

t

2nd Cycle L

-*

L

t

of contamination, because there were eight cycles of Edman of the bound ATPmolecule due to the subsequent binding degradation before this step. In other words, if there were any AMP. Fig. 7A shows that the fluorescence emission of ADATP-labeled adenylate kinase is not affected by ATP added contaminating amino acids or peptides in the sample, they should have been pickedup by previous cycles. The possibility to the medium, but is enhanced by AMP. This is a special that His-36 was labeled is qualitatively consistent with the case of the general method of monitoringconformation observation of diminished His peak, the appearance of unde- change at thecatalytic site of an enzyme by an in situ fined peaks, and the detection of radioactivity in this remain- fluorescence probe. The predicted theoretical relationship( f ing fragment of P4. Thus we may identify P1 with Pro-Thr- - f i ) / ( f 2 - f) = [AMP]/KAMp(15) between the fluorescence Cys*-Glu-Lys-Ile-Val-His-Lys-intensity f and AMP concentration is verified by the linear Leu-Leu*-Leu andP4with plot in Fig. 7B which also gives a dissociation constant of Tyr-Gly-Tyr-Thr-His*, where the asterisks indicate theposiKAMP = 0.24 mM for the labeled enzyme. tions of radioactive label. These results are in full agreement The observation that ATP has effect no on thefluorescence with the conclusions from x-ray diffraction (18) and NMR of the AD-ATP label supports the previous conclusion that (19) studies and are not inconsistent with the recent x-ray ATP is not bound at the AMP siteof adenylate kinase (17). data on other adenylate kinases in the absence of substrate The observed enhancement of the fluorescence of AD-ATP(8,20). The residues Cys-25, His-36, and Leu-115 are all very labeled enzyme by AMP indicates that the bindingof AMP close to the adenine group of bound ATP according to the at the AMP site causes environmental change at the ATP model proposed by Fry et al. (19). site. Fluorometric Data-The present affinity label provides an DISCUSSION additional piece of structural information on adenylate kinase which has not been obtained from x-ray diffraction and NMR The photoreactive ATP analogue ['TIAD-ATP with its data, Le. the environmental change in theimmediate vicinity environment-sensitive fluorescent dansyl groupmay be a

7986

A Fluorescent PhotoaffinityReagent

IY b-

t

fl

6th

h

K

7th

f

FIG. 6. Analysis and sequencing of radioactive peptide P4 from [“C]

AD-ATP-labeled adenylate kinase. The procedures for determining the amino acid composition and sequence of P4 are similar to those for P1 in Fig. 5. Since no fluorescent OPA derivative was obtained in the first Edman degradation cycle, this step was repeated with Scarboxymethylated peptide P2 (see Table I). One-letter amino acid code is used.

A

-

A

t

J Y Analysis of

P4 after 8th

f

H

useful tool for studying many ATP-binding proteins. Upon illumination by UV light, a nitrene and ahighly energetic Nz are produced. Because the molecular weight of this nitrene (781.5) is much larger than that of the departing Nf, only 3.5% of the -130 kcal/mol available energy (equal to the energy of 280 nm light plus released chemical energy) or -4.5 kcal/mol was initially received by the nitrene because of the conservation of momentum. Consequently, the highly reactive nitrene cannot escape from the active site andmay label only those groups at or near the site. Quantitatively the number of amino acid residues that are chemically altered by reaction with the nitrene or collision with the highly energetic NZ (Table I) may belarger than thenumber of labels that become covalently attached to these residues (Fig. 4A).The resulting covalently attached radioactive label can be used to identify the amino acid residues at the active site, and the environment-sensitive dansyl group can be used as an in situ probe

for monitoring effector- or protein phosphorylation-induced conformation change at theactive site. The hydrophobic dansyl group in AD-ATP could make this analogue bind to theactive site of many kinases and ATPases with even greater affinity than ATP itself. In the particular case of adenylate kinase, KI is 10’- or lo3-fold lower than the K, for ATP. This observation suggests the possible use of ADATP as aselective photoreagent for inhibiting many undesirable processes in the cell. The identification of Leu-115, Cys25, and probably His-36 as theamino acid residues selectively labeled by AD-ATP at the active site of adenylate kinase is also consistent with the datafrom x-ray diffraction and NMR studies. These results give us a reasonable degree of confidence in using AD-ATP for studying otherATP-binding proteins which may havetoo high a molecular weight or may be unavailable in sufficient amounts for x-ray diffraction and NMR studies.

7987

A Fluorescent Photoaffinity Reagent TABLE I1 Sequencing of ['4C]AD-ATP-labeledpeptides P1 and P4 from rabbit muscle adenylate kinase by Edman degradation The amino acid residues expected are based on the sequences listed in the legend to Table I. Residue Peptide

Cycle

1

2 3 4 5 P4 Ile His

1 2 3 4 5 6

Pro" Thr Leu Leu Leu

Pro" Thr Leu Leu Leu

120 146 138