NUCLEOPHILIC REACTIONS OF ACYLPHOSPHATES
Nov. 5 , 1061
of a proton froin water to the carbonyl oxygen atom. The rate constants for general base catalysis of acetylimidazole hydrolysis by water and by imidazole according to eq. 6 rate = [ k I m ] Z i k ~ i [ B i ]
(6 )
are 9.0 X 10-5 and 0.14 1. mole-' min.-', respectively. This gives a Bronsted slope, p, of 0.37, which is similar to the value of 0.45 observed for general base catalysis of ester hydrolysis. The hydrolysis of AcMeIm+ is also subject t o general base catalysis by N-methylimidazole. This catalysis must be classical general base catalysis, because nucleophilic displacement would only regenerate starting material and the rate of Nmethylimidazole-catalyzed hydrolysis is decreased in solvent DzO. The existence of general base catalysis of this reaction is somewhat unexpected, since the relative importance of general base catalysis decreases as the leaving group becomes better in the aminolysis of substituted phenyl acetates, and the reactions of 0-nitrophenyl acetate, which has a leaving group with a pK of 7, are generally not detectably subject to classical general base catalysis.1 1 , 1 2 The pK of N-methylimidazole, the
~CONTRIBUTIOK KO.124 FROM
THE GRADCATE DEPARTMEXT OF
4393
leaving group of XcRleIm+, is also 7 and the rates of its reactions are several orders of magnitude greater than those of p-nitrophenyl acetate, yet general base catalysis makes an important contribution to the observed rates of reaction of this compound. The methyl group on the imidazole moiety of AcMeIm' reduces the ambiguity in respect to possible mechanisms for this general base catalysis,1° as discussed above, and leaves V and VI as reasonable transition states, in which, again, the 0-C bond may be partly or fully formed. I--\
V I
VI (11) W P. Jencks and J. Carriuolo, J . Am. Chem. SOC.,83, G75 (1960). (12) T . C. Bruice and h l . F. hfayahi, ibid., 82, 30G7
(19GO).
BIOCHEMISTRY, BRANDEIS UNIVERSITY, WALTHAM 54, MASS.]
Mechanism and Catalysis of Reactions of Acyl Phosphates. I. Nucleophilic Reactions1 BY GIOVANNI DI SABATO AND WILLIAM P.
JENCKS
RECEIVEDJUNE 14, 1961 Acyl transfer reactions from acetyl phosphatc t o n-butylamine, ammonia, glycine and hydroxylamine are subject t o general base catalysis, as shown by a greater than first-order dependence of the rate on the concentration of amine buffers. The reactions of acetyl phosphate at neutral pH with hydroxylamine, aniline, morpholine, N-methylimidazole, glycine and glycylglycine give C-0 bond breaking and proceed predominantly through a n acid-catalyzed pathway; i.e., they involve a reaction of amine base with the acetyl phosphate monoanion. The existence of these catalyzed reaction paths partially accounts for the relatively high reactivity of acetyl phosphate. The reaction with glycine and the neutral and base-catalyzed hydrolysis of acetyl phosphate are catalyzed by calcium ion. The reactions with pyridine, 4methylpyridine, triethylenediamine and probably trimethylamine give P-0 bond breaking; in the presence of fluoride, fluorophosphate is formed, suggesting t h a t these reactions represent nucleophilic catalysis of phosphoryl transfer. Fluoride also reacts directly with the acetyl phosphate monoanion t o give fluorophosphate, but does not react appreciably with the acetyl phosphate dianion.
The high reactivity of acetylimidazole at neutral
pH is due not so much to a high reactivity of the compound itself as to the availability of facile acidand general base-catalyzed paths for its reactions % The present study was undertaken to determine the extent to which the reactions of acetyl phosphate, another "energy-rich" compound of some biochemical importance,3--6 proceed by similar catalytic pathways in aqueous solution near neutrality. In the course of the study several unexpected characteristics of acyl phosphate reactions were encountered, (1) Supported in part by the National Cancer Institute of the N a tional Institutes of Health (Grant C-SO75 and Training G r a n t C R T 5033) a n d the National Science Foundation. For a preliminary report, see Abstracts, 137th Meeting, American Chemical Society, Cleveland, Ohio, 1960,p. 77-0. (2) W. P. Jencks a n d J. Carriuolo, J . Bioi. Cliem., 234, 1272, 1280
(1959). (3) F. Lynen, Ber., 7 3 , 367 (1940). (4) F. Lipmann and L. C . Tuttle, J . B i d . Chem., 153, 571 (1914). ( 5 ) D. E. Koshland, Jr., J . A m . Chem. Soc., 73, 4103 (1951). ( 0 ) 1). E. Koshland, Tr., rbrd., 74, 2288 (1Y52).
Experimental Dilithium acetyl phosphate was prepared by the procedure of A ~ i s o n . ~ Acetyl phenyl phosphate was prepared by a modification of this Other reagents, except for reagent grade inorganic salts, were recrystallized or redistilled before use. Water and deuterium oxide were glass-distilled. The reactions were started by adding a freshly prcpared solution of acyl phosphate to a reaction mixture a t 39.0" which contained a large excess of the other reactant under investigation. Aliquots were withdrawn at appropriate time intervals and analyzed for remaining acyl phosphate by conversion to hydroxamic acid.8 The extent of the reaction, x m- x t , was plotted on semi-logarithmic graph paper and a pseudo-first-order rate constant obtained from the half-time, using the formula k = 0.693/t1/~. Five to ten points were used for the determination of each first-order constant. The observed rate constants were corrected for rates of hydrolysis, determined separately at the same p H and ionic strength. Second-order rate constants were obtained from the slopes of plots of the observed first-order constants against the concentration of the second reactant. Catalytic constants were obtained from the slope of plots of the appropriate observed first- or second-order con(7) A W. D. Avison, J . Chem. Soc.. 732 (1955). (8) P. Liptuann and L. C. Tuttle, J . Bioi. Chem., 169, 21 (1945).
GIOVANNI DI SABATO AND S . T . T P. ~ ~JBNCKS ~ ~ ~ ~ ~ ~
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150 100
0.4 0.6M ISH] free base. Fig. 1.-The effect of ainniotiia concentration cn the anirnonolysis of acetyl phosphate a t pH 9.5, 39.0" and ionic strength 0.6: solid line, rate for the catalyzed plus uncatalyzed reaction, calculated from the rate constants of Table I ; dashed line, calculated rate for the uticatalyzed reaction; upper line, observed second-order rate coiistants as a function of ammonia concentration.
0.2
stants against the concentration of the catalyst. Iieactions leading to the disappearance of thiols were carried M ethylenediaminetetraacetic acid in the reout with action mixtures and were followed by measuring the disappearance of the free thiol group by the nitroprusside method.g For calcium-catalyzed reactions, excess ethylenediaminetetraacetic acid was added to the assay mixtures t o prevent calcium carbonate precipitation. Since the thiol was not present in excess, complex kinetics, between zero and first order, were obtained in these experiments and no attempt \vas made t o calculate rate constants. Inorganic phosphate release 011 the reactioii of acetyl phosphate with tertiary amines iii methanol was determined by the Fiske and SubbaRow Inethod'O after precipitation as the calcium salt.4 Fluorophosphate was determined as the difference in inorganic phosphate before and after hydrolysis in 1 111 HC1 for 10 minutes a t 100'. Reaction mixtures containing fluoride were incubated in polyethylcne tubes. Fluoride and triethylenediamine were shown not t o interfere with the phosphate analyses after dilution, although more concentratecl tertiary amines do cause interference. Fluorciphosphate was shown to be stable under the conditions r i f incubation of the reaction mixtures, except a t acid ,pH. I'luorophosphatc: was identified by paper electrophoresis o r 1 Whatnian $31 double thickuess paper in 0.02 Jf sodiuiti citrate-0.003 X citric acid and in 0.08 Jf sodium acetate0.02 A 1 acctic acid buffers with a potential of 10 v./cm. for 4 hours a t 3-5". For these experiments, the reactions were run with 0.4 f l f acetyl phosphate and 2 111 fluoride, so that the ionic strength would not be excessive after dilution t o give 0.2-0.3 ~mole/0.02-ml. aliquot. The dried sheets wcre sprayed with molybdate mixture," dried at 65" for 1 hour, sprayed with 1Tc SiiClz in 1 M HCI, and exposed to ammonia vapors; niigratiou in cm.: fluorophosphate, (9) H. R. Mahler, S. J. Wakil a n d R. 31. Bock, J . Diol. Chein., 204, 453 ( 1 0 5 3 ) . (10) C. 1%.Fiske a n d Y.SubbaRow, ibid., 66, 373 (li725,) 111) C S. Hanes a n d F. A . Ishrrwood, S a t w e , 1 6 4 , 1107 (IDD)
Vol. s3
pyrophosphate and phosphate, 30, 25.5, 20 (citrate buffer) and 24,21.5, 18 (acetate buffer), respectively. The reaction product moved a t the same rate as known fluorophosphate and did not separate from fluorophospliate added t o t h e reaction mixtures. The reactioii of liydroxylaniinc with acetyl phosphate, as with other activated acyl groups, gives both S-acylation (hydroxaniic acid) atid 0-acylation (0-acetylhydroxylamine) in the initial reaction, which is followed by a slower, hydroxylamine-dependent, isomerization of the O-acetylhydroxylamine to hydroxaniic acid.lZ At pH 4.1 and 6.4, hydroxaniic acid formation accouiits for 74-iiyo of the iiiitial reaction; this is f d o m e d by a slower formation of hydroxaniic acid at a rate equal t o the rate of isouierization of 0acetylhydroxylamine. The ratio of 0 to S acylation was riot affected by variation of the liydrosylarnitic concentration from 0.1 to 0.5 df at PH 6.4. The rate constants reported here were obtained from the iuitial first-order forination of hydrosainic acid and were inultiplicd by 0.75 to givc the rates of reactioii of the liydroxylmiiiie nitrugeii atom.
Results The rate of the reaction of acetyl phosphate with ammonia a t pH 9.3 increases more rapidly than the first power of the ammonia concentration a t constant pH and ionic strength (Fig. 1). This is evidence for general base catalysis of the reaction by a second molecule of ammonia, as previously demonstrated for the reaction of phenyl acetate with a number of amines and of ethyl formate with wbutylamine. 13,14 The rate constants for the catalyzed and uncatalyzed reactions (Table I) were calculated as previously describe~l'~ according to the rate law LI
kz[-i~P-][?u"a]
-fks[Al~P=][?;H3/2 (1)
and the solid line in Fig. 1 was drawn froin the rate constants so obtained. The rate of the reaction with 0.1 and 0.2 -11ammonia a t pH 9.4 was found to be unchanged on variation of the ionic strength from 0.G to 1.0 by addition of KC1. Thc reactions of acetyl phosphate with n-butylamine and glycine were studied in the same manner and also were found to be subject to general base catalysis (Table I). As in the case of phenyl acetate, and presumably for the same reason^,'^ no such catalysis was found of the reactions with piperidine, trimethylamine, morpholine and triethylenediamine (diazobicyclooctane) . The reaction with hydroxylamine shows a-".I rate maximum as well as a term second-order in respect to hydroxylamine; the rates a t pH 6.5 and flH 7.0 are entirely accounted for by the rate law =
k~'[.lcP-][SH?OH]$- ks'[AcP-][NH20H]'
(la)
This is the only compound for which such a catalyzed reaction of the acetyl phosphate monoanion was detected. The rate of the reactioii of ariiline with acetyl phosphate shows a inaxiiiiuin a t PH 4.7 and. is very sniall a t flH 7 (Fig. 2). Such a pH-rate niaxiniutn is consistent with a reaction of the acetyl phosphate dianion (dcP=) with the conjugate acid of aniline 'o'=ki"[.-ZcP=][KSH3+]
(2)
or a kinetically indistinguishable reaction of the acetyl phosphate monoanion (AcP-) with free aniline i' =
k2'[AcP ][lI".il
(3)
(12) 15'. P. Jrncks, J . A m Chc?ii. S o / . , 80, 4,5Sl, 4385 (lCJ58). (13) W. P. Jencks a n d J. Carriuolo, ibid.,82, 675 ( 1 9 6 0 ) . (1-4) J F.B u n n e t t a n d G . T. Ilavis, iDi
