ARCEIVES OF LIIOCHEMISTIlY AND 13IOPHYSICL 143, 340-341 (1971)

The Potential of CarboxyliePhosphoric Mixed Anhydrides as Specific Reogents for Enzymic

Binding Sites for Alkyl Phosphates. Inactivation of an Enzyme by an Anhydride

lsosteric with Adenosine 5’.Phosphate’

Alkyl phosphates (e.g., I, Iti = sugar or nucleo- aide residue, R* = H, phosphoryl, pyrophosphoryl, sugar phosphoryl, or nucleotidyl) are substrates or allosteric effectow for numerous enzymes, whereas the corresponding primary alcohols (R.*CHnOH) are usually devoid of such properties. Mechanisms by which t,he phosphate portion interacts with the enzymes could be elucidated with alkyl phosphale analogs which are sufficiently isosteric to retain selective binding to the sub- strate site and which could bond covalently to an amino acid in t,hat. site. Studies wit.h purine nucleotides (l-3) showed that small electronic or steric variations in atoms bonded to the phos- phorus frequently reduce or eliminate selective binding of these alkyl phosphates to substrate sites. The desirability of retaining an unmodified phosphate moiety to assure strong binding sug- gested that promising analogs of the required type might be carboxylic-phosphoric mixed an- hydrides, II, which are not only closely isosteric with I but which iu the analog-enzyme complex could be subject to nucleophilic attack at two al- ternative centers, i.e., t.he carbonyl carbon and also the thermodynamically unfavored phosphorus stom. This communication describes synthesis of the hitherto unknown auhydride III isosteric with AMP and presents evidence that it is a specific and exceptionally powerful inact,ivator of an AMP-utiliring enzyme.

The synthesis was based on an anion-exchange method (4, 5) for couvemion of 5’-nucleotides to phosphoanhydride derivat.ives. Dioxane, X,h’- dimethylformamide and diphenylphosphorochlori- date (0.48 mmole) were added t.o the sodium salt of 9-@-n-ribofuranosyluronic acid)adenine (6) (0.5 mmole). The suspension was stirred for 5 hr, filtered from sodium chloride and unreacted ___, ._-----.~ ---. -..-. --~

1 The work was support,ed by U.S.P.H.S. grants CA-11196, CA-66927, and RR-05539, Ameri- can Cancer Society grants IN-49 and T-490, an award from the Pennsylvania Science and En- gineering ‘Fund, and an appropriat*ion from the Commonwealth of Pennsylvania. We thank Dr. L. II. Cohen for providing the lyase and its sub- strate.

sodium carboxylate (tot;al, 85 mg), and light pet,roleum-diethyl ether was added to the fi1trat.e to precipitat,c the P, P-diphenyl est,er of III. This material was reprecipitat.ed five times by additiou of et.her to its solut,ion in dioxane to give a white powder (0.27-0.29 g, 11&118%) which contained traces of dioxane and N,N-dimethylformamidr not readily removed in vaczlo or by further precipi- tations; ir 3250, 1748, 1642, 1585, 1483, 1210, 1100~ 916, 782, 760, and 692 cm-l. With anhydrous am monia-N,N-dimet.hylformamide the product, gave solely diphenylphosphoric acid (identified by i P and tic* in systems A and I)) and the amide of i he st.arting acid (described below). The diphenyl est.er of III was dissolved immediately in dioxane and to t,his was added a pyridine solut,ion of mono(tri-n-b~~t,ylammonir~m) phosphate (1 mmole) and t.ri-n-butylamine (1 mmole). The solution was stored at 22” until precipitated samples lacked mouosubstit,uted phenyl absorp- tion at, 782, 760, and 690 cm-l (2- t,o 6-hr reaction). The tri-n-butylammonium salt of III was repre- cipitated zur described for its diphenyl ester and obtained as a whit.e powder (0.28-0.31 g; 8090% yield calculaled from IIV absorption) containing ca. 0.5 molar eqniv. of tri-n-butylammonium phosphate; ir 3250, 315@h, 1742, 1642, 1602sh, 1200, and 1090 cm-‘; uv spectra were ident,ical wit,h those of the st.art,ing acid (6). The unstable material was preserved as a 1% solution in dry dimethylformamide at -25” under nitrogen; after several days, uv spectral changes suggestive of NG-acylaminopurine nucleoside formation were det.ectable. Treatment of III with dry am- monia-NJ’-dimet hylformamide gave the car- boxamide of the starting material as the only ultraviolet,-absorbing product (t.lc systems A, B, and C); after isolation by preparative tic (sysi.em A) it formed needles, mp 275’ dec, from methanol. Anal. Calcd for CluHraNsO~: C, 42.86; H, 4.32; -~ - ..--- - ~-

2 On cellulose; system A, ethanol-l I am- monium acet,at.e (7:3); B, 2-propanol-ammonia- water (7:1:2); C, n-butanol-aoet.ic acid-water (4:1:5); I), 2-propanol-dimethylformamide--

methyl et.hyl ketone-water-ammonia (40:40:40: - 78:2).

340

COMMUNICATIONS 341

0 00

R~CH*Ob(OH)(OR~j R1 t: ’ OI$OH)(ORa)

I II

OH OH

III

Fro. 1

N, 29.99. Found: C, 43.04; H, 4.23; N, 30.08; ir 1689 cm-1 (C=O) ; uv max (pH 3) 258 rnp (e 14,3OOj, (pH 7) 259 (14,300).

The solution of III was mixed at. 22” with p:trt,ially purified E. coli adenylosuccinate lyasc (EC 4.3.2.2) in 40 mM Tris chloride-10 rnM so- dium ethylenediamine tetraacetat,e (pH 7.7) to give 80 PM III (and 40 p.~ tri-n-butyiammonium phosphate); ca. 5 set later ammonium adenylo- succinate (finally 120 m) was added. Uv absorb- ance measurements (7) showed 95-99% reduction in the rate (0.3 mpmoles/min) of formation of AMP. This inhibition was abolished (a) by 30-set hydrolysis of III in the buffer prior to successive addit.ion of enzyme and substrale, (b) by prior addition to the enzyme of 120~~ adeny1osuccinat.e (Ii,,, = 20~~)~ and (c) by substituting 1 rnb1 tri-n- butylammonium phosphate or 2 111M acetyl phox- phatte (20 min interaction with enzyme) for III. T.ess than 10% of the acetyl phosphate would have undergone hydrolysis during this relat ivel) Icngt.hy period (8), and t,he lack of reversible inhibition by this compound sr1ggest.s that its inability to effect, inactivation may arise from IJoOr affinity for the nucleot.ide binding sit.e. On I he other hand, AMP and adenylosuccinate c0mpet.e for the same sit,e of a yeast lyttse (9) and on t,he assumption that this is (rue for the E. coli lyaee t.he present findings tend to indicate that III ncylat.es or phosphorylat,es the enzyme near t,he site at. which the phosphate moiety of AMP makes its contribution (9) to AMP binding.

The acyl phosphate III hydrolyzes at least 100 f irnes faster than acet.yl phosphate at pH 7.7 (8),

thus suggesting intramolecular assistance from the favorably located 3 nit,rogen or the 3’ hydroxyl. Noninvolvement of the 3’ hydroxyl is suggested by the finding that hydrolysis of III did not. furnish the 3’.phosphate of the adenosine 5’- carboxylic acid; on the other hand, t,he hydrolysis might involve a 3-acylated intermediate because 3,5’-cycloadenosiues do form with great facility (10) and because pyridine-catalyzed hydrolysis of acetic anhydride proceeds via :V-acetylpyridinium ions (11).

The anion-exchange met.hod is general for the synthesis of nucleotide anhydrides (5) and in view of the successful synt,hesis of III should be adapt.- able to other phosphoric-carboxylic anhydrides, particularly since many of these (e.g., the isosteres of pyrimidine and pyridine coenzymes) lack the equivalent of N-3 of AMP and hence are expected IO be hydrolytically more stable than III.

Compound III showed lit.tle or no tendency to inact.ivate AMP deaminase of rabbit, muscle. Syntheses of addit ional phosphoric-carboxylic mixed anhydrides isost.eric with metabolically import,ant alkyd phosphates and enzyme studies with them will bc detailed later.

ALEXANDER HAMPTON

PETEN .J. H.\arw

Yhe Inslilute joor Cancer Research Fox Chase Philadelph.ia, Pennsylvania 19111 Received December 16, 1970

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