4699 and as determined experimentally. Plots of t(R, - R o b J d ) us. R o b s d for each kinetic experiment are of excellent linearity, and the rate constants as calculated by this method are summarized in Table I. A detailed kinetic study was then made under conditions in which one of the reactants in each experiment was in sufficient excess to ensure greater than 99.5 % neutralization of the minor component. As a n example, for neutralization of 1-phenyl-1-nitroethane to be 99.5% complete under the kinetic conditions (eq 7), n must be 1.4; for I I = 10, neutralization occurs to almost 100%. For use of eq 7 effectively, R, of accuracy is required. It was more reliable and convenient to use excess nitro compound than excess hydroxide ion in each kinetic experiment. Excess hydroxide ion did not lead to a totally constant R, value in any system; with time R, as measured experimentally increased slowly. Excess hydroxide ion could be used reliably upon measuring R, at times corresponding to 8-10 half-lives for neutralization. It was more convenient to use excess nitro compound ( n = 1.4-10) because R , stabilized more satisfactorily by this method; upon prolonged storage of the kinetic solutions the resistance of a neutralized solution began to drop slowly but this did not lead to serious inconvenience or experimental complication. A further advantage in using excess nitro compound is that the resistance change is larger than when the base is the reactant in excess. This technique also reduces the effect of any error introduced by approximation of Ro. The rate constants and the kinetic parameters for neutralization of the nitro compounds by this method are summarized in Table I. Infrared Spectra of Alkanenitronates. Nitromethane, nitroethane, and 2-nitropropane were fractionated and stored over Linde 5A Molecular Sieves. The pure nitroalkanes (1.0 g) were dissolved in pentane (25 ml) and treated, respectively, with 0.5 equiv of butyllithium (Foote Chemical Co., 1 M i n hexane), sodium methoxide powder (sublimed), and potassium t-butoxide powder (MSA Research Corp.). The salts precipitated almost immediately and the mixtures began refluxing. The slurries were stirred for 5
min, filtered, washed with fresh pentane (3 X 25 ml), and vacuum dried. 3 The white salts were ground with anhydrous potassium bromide crystals (Fisher Chemical Co.) and pressed under vacuum in a pellet die to give clear, transparent wafers. The infrared spectra of each salt were immediately determined on a Perkin-Elmer Infracord spectrophotometer. This position of the major band near 1650 cm-1 is reported in Table VI. Ultraviolet Spectra of Alkanenitronates. For determining the wavelengths ( x +. x * ) of maximum absorption of the various alkanenitronates (Tables V and VII), each nitro compound was treated with excess sodium methoxide or pure sodium salts of the nitronates were dissolved in the various hydrogen bonding or aprotic solvents to give alkaline solutions approximately 10-4 M in nitro compound. The solutions were always blanketed with nitrogen and analyzed within 0.25 hr after preparation. Cary (Model 14) and Beckman (DU) spectrophotometers were used for the measurements. For the data in Table IV and Figure 7, solutions from kinetic runs at 0" were used in which the initial concentrations of the reactants were the same. After the resistance of a solution had reached a constant or a maximum value, a n aliquot was taken and diluted with 50% (vol) dioxane-water so that the final concentration of the nitronate was -7.5 X M. The spectra of the anions were determined immediately with a Beckmann (DU) spectrophotometer. The extinction coefficients are only approximate since neutralization was not complete (>94%) and there was no correction for hydrolysis of the spectral samples upon dilution. Use of excess sodium hydroxide resulted in solutions whose absorptions were time dependent in which the extinction coefficients became less. (34) Methanenitronates are very dangerous and were used as slightly damp powders.
Models of Ribonuclease Action. 11. Specific Acid, Specific Base, and Neutral Pathways for Hydrolysis of a Nucleotide Diester Analog'" D. A. Usher,3 David I. Richardson, Jr.,4 and D. G . Oakenfulls Contribution f r o m the Department of Chemistry, Cornell Uniuersity, Ithaca, New York 14850. Receiued September 18, 1969 Abstract: T h e rate of production of phenol f r o m the phenyl ester I h a s been measured a s a function of p H at 50" a n d ionic strength 0.1. T h e cyclic phosphate JII is the soleinitial product a t pH values a b o v e 4 , a n d it is considered likely t h a t this is also true f o r the reaction in acid. F o u r kinetically distinct terms appear in the rate equation: kl(HEH)(H); k2(EH)(H); k3(EH); k4(EH)(OH), a n d the four rate constants a n d the acid dissociation constant of t h e phosphate were obtained by a weighted nonlinear least squares analysis. A large specific salt effect was shown by k4. The kinetic pK, of the neighboring hydroxyl group of I h a s been measured by stopped-flow kinetics i n strong base. Plausible mechanisms for these reactions are considered, a n d i t is shown t h a t these results can b e useful in considering some of t h e elementary steps of ribonuclease action.
ince 1920, when Jones recorded6 in the American Journal of Physiology his recent discovery that a boiled aqueous extract of pig pancreas was able to
S
(1) This research was supported by the National Institutes of Health under Grant GM 13335. (2) A preliminary report of a part of this work has appeared: D. G. Oakenfull, D. I. Richardson, Jr., and D. A. Usher, J . Amer. Chem. Soc., 89, 5491 (1967). (3) To whom enquiries regarding this paper should be addressed: Career Development Awardee of the National Institutes of Health (l-K4-GM-42,407). (4) Predoctoral N. I. H. Trainee, 2101-GM00834. ( 5 ) . Postdoctoral N. I. H. Trainee, 2T01-GM00834; Division of Food Preservation, C.S.I.R.O., Ryde N. S. W., Australia. (6) W. Jones, Amer. J . Physiol., 52, 203 (1920).
hydrolyze yeast nucleic acid, ribonucleases from many different sources have come under the close scrutiny of a large number of workers in s e v e r a l disciplines, and today one of the most studied of all enzymes is a member of this group.' Enormous progress has been made s i n c e the first crystallizations of b o v i n e pancreatic ribonuclease in 1940. (7) For reviews, see E. A. Barnard, Ann. Rev. Biochem., 38,677 (1969); H. A. Scheraga, Fed. Proc., 26, 1380 (1967); J. P. Hummel and G. Kalnitsky, Ann. Rea. Biochem., 33, 15 (1964); H. Witzel, Progr. Nucleic Acid Res., 2 , 221 (1963); F . H. Westheimer, Aduan. Enzymol., 24, 441 (1962); H. A. Scheraga and J . A. Rupley, ibid., 24, 161 (1962); C. A. Dekker, Ann. Reu. Biochem., 29, 453 (1960); D. M. Brown and Sir A. R. Todd, ibid., 24, 311 (1955).
Usher, Richardson, Oakenfull
1 Models of'Ribonuclease Action
4700
Among the important advances are determination of the specificity and mode of action of the e n ~ y m eiden,~ -'* tification of the residues at the active ~ i t e , ~ , ~ determination of the complete primary sequenceI5 and three dimensional structure, 16, l7 direct study of the imidazoleC-2 protons of the four histidine residues by nmr spectroscopy, detailed steady-state71l9 and transient state kinetic analyses, l 9 conversion to RNase-S20 and production of partially synthetic enzyme,21 and now the first fully synthetic enzyme.?? At many stages along the way, suggestions of mechanism have been ad~anced.~ It is well established that RNase catalyzes the breakdown of a ribonucleic acid by two steps: a transesterification followed by hydrolysis of the resulting cyclic phosphate. Thus, it is surprising that although there have been many investigations of the lability conferred on a phosphate diester by the presence of a neighboring hydroxyl group,23 no single model of this first reaction that is catalyzed by the enzyme has ever been analyzed in detail. Certainly our knowledge of this reaction is more sketchy than that of the hydrolysis of carboxylic esters. Part of the reason for this may be the slowness of the nonenzymic reaction at other than extremes of pH; extrapolation to pH 7 of the secondorder rate constant for the hydroxide ion catalyzed hydrolysis of ~ytidylylcytidine,~~ assuming no incursion of neutral or acid-catalyzed paths, predicts a half-life for this reaction of about l o 3 to lo4 days, even at 60". Indeed, the cyclic phosphate diesters, though remarkable for their ease of hydrolysis in both acid and base, 2 j have half-lives of a similar order of magnitude around neutrality at 25 '. In view of the importance of an understanding of the mechanism of participation by neighboring hydroxyl i n the hydrolysis of both ribonucleic acids and phospholipids, and since a control reaction was required for our studies of synthetic catalysts, we decided to investigate in detail the hydrolysis of a model of a dinucleoside (8) M. Kunitz, J . Gerz. Physiol., 24, 15 (1940); M. R. McDonald, ibid., 32, 33, 39 (1948). (9) L. Weil and T. S. Seibles, Arch. Biochem. Biophys., 54,368 (1955). (10) H . G. Gundlach, W. H. Stein, and S. Moore, J . Biol. Chem., 23,
1754 (1959). (11) E. A. Barnard and W. D. Stein, J . Mol. Biol., 1 , 339 (1959). (12) D. Findlay, D. G. Herries, A. P. Mathias, B. R. Rabin, and C. A. Ross. Nature. 190. 781 (1961). ( l j ) A. M . ~Crestfield,' W. H. Stein, and S. Moore, J . B i d . Chent., 238, 2413, 2421 (1963). (14) C. H. W. Hirs, Brookhaoen S y m p . Biol., No. 15, 154 (1962). (15) D. G. Smyth, W. H. Stein, and S. Moore, J . Biol. Chem., 238, 227 (1963). (16) G. Kartha, J. Bello, and D. Harker, Narure, 213, 862 (1967). (17) H. W. Wyckoff, I> k-5 and k5 became rate determining. Since the reaction of EH' with an amine base such as morpholine would be thermodynamically downhill, this alternative route for deprotonation of the intermediate should have a rate constant of about 1010 M-' sec-','j3 and observed rates of the order of k5 should be attainable even at pH 8 say, by the addition of 1.0 M 1 : l amine buffer. This is not found, and argues against this mechanism, at least in this simplified form. A mechanism involving hydroxide as a general base is similar to the mechanism of Scheme I but would require either a concerted reaction, which is probably not consistent with the deuterium solvent kinetic isotope effect (see above), or that the ring closure be more rapid than reprotonation of the alk~ latter condition oxide anion (about 1O'O ~ e c - ' ) . ~The clearly does not hold, as it would imply that observed rates of this same magnitude could be achieved in 1 M
stant for deprotonation of EH'j3 is likely to exceed the first-order constant for ring closure ( k , in Scheme I) down to just below pH 5 . It appears likely then that the major path is as shown in Scheme I and involves an "in-line" s N 2 type of displacement of phenoxide ion by the neighboring alkoxide. There is no evidence for a pentacoordinate intermediate in this reaction, the lack of base-catalyzed migration of an alkyl phosphoryl group to a neighboring hydroxyl64does not, however, exclude such a species, since the pseudorotation that would be required for such a migration would be expected to be inhibited by the preference of a P-0- group to maintain a basal position in the trigonal bi~yramid.65-~~ (64) D. M. Brown, D. I. Magrath, A. H. Neilson, and A. R. Todd, Nature, 177, 1124 (1956); D. M. Brown and D. A. Usher, J . Chem. SOC.,6547 (1965). (65) D.A. Usher, Proc. Nat. Acad. Sci. U.S.,62, 661 (1969). (66) R. Kluger, F. Covitz, E. Dennis, L. D. Williams, and F. H. Westheimer, J . Amer. Chem. SOC.,91,6066 (1969). (67) It is possible that the rate of decomposition of a dianionic pentacoordinate intermediate would exceed the rate of its protonation by water. The dependence on pH of the product ratio for hydrolysis of 12, /
I
HO
EH
(63) M. Eigen, Angew. Chem. Intern. Ed. Eng., 3, l(1964).
breakdown
2 pseudorotation (0-' as pivot)
E'
base. If there were an intermediate (E') it would be expected to partition in favor of product formation (ks> Li)so that kobsdshould approximate to the value of ki. Since the experimental rate constant (7.0 M-' sec-I) is much less than that expected for removal of a proton from E H by hydroxide, it follows that, intermediate or not, a preequilibrium ionization must exist, as in Scheme I. At 25" the pseudo-first-order rate con-
Including the point at
k,
breakdown
/
0-
>
pseudorotat ion (0- as pivot)
methyl ethylene phosphate56 is consistent with ka > kb for the monoanion and kd 3 ke for the dianion. In the latter case the pseudorotation is aided by the PO- group moving from an apical to a basal position. The rate of breakdown of the dianion is likely to exceed that of the
Usher, Richardson, Oakenfull 1 Models of Ribonuclease Action
4708 The calculated second-order rate constant for hy(111) in base is about 1000 times slower than the ring droxide ion catalysis (k4 = 7.0 M-1 sec-I at SO') thus closure reaction of represents a combination of rate and equilibrium conThe Neutral Pathway (pH 4-61, In contrast t o the base- and the acid-catalyzed reactions of I, there has stants (kK,/Kw) and since Kz is known only at 25.3" in water at an ionic strength of 1, activation parameters been no previous clear demonstration of a neutral rate are not simply interpretable. However, for the purfor any 2-hydroxy phosphate diester. Similar restraints on the mechanism operate here as with the base-cataposes of discussion it will be assumed that the value of lyzed reaction, since the cyclic phosphate and phenol K2 at 50" and ionic strength 0.1 is about 10-13.2,thus giving it a temperature dependence similar to that of are the sole initial products. A kinetic equivalent to Kw.53 The value of k at 50" would then be (Kwk4/K2)E the reaction (EH) (c) is the process d ; either of these 6 sec-I. The hydroxide ion catalyzed cyclization of a two mechanisms (and general base or general acid varseries of esters of propane-I ,2-diol monophosphate iants of them)27could be shown as including a pentagave70a linear plot of log koH (80") us. the pK, ( 2 5 " ) of coordinate intermediate. Reaction cia (c) with a the conjugate acid of the leaving group that had a grasec-I at 50" would imply specific rate of 4.53 X dient of 0.56; combination of the present result with that that deprotonation of the neighboring hydroxyl group of Cox and C l e ~ e l a n d ' for ~ the methyl ester of tetraincreases the rate of nucleophilic attack on phosphorus hydrofurandiol phosphate defines a line with a graby a factor of about IO7. If we assume K2 = dient of 0.59 (at SO0), though the second-order rate constants are about 1000 times faster than for the propanediol phosphate esters at the same temperature. The rate of hydrolysis of uridylyl(3'-5')adenosineZ4 in base is within a factor of 2 of the rate of the methyl ester of tetrahydrofuran diol ph0sphate.~1 In both reactions the leaving group is a primary alkoxide anion, thus the more complicated structure of the dinucleoside phosphate has little effect on the rate of reaction in base. However, the ring oxygen has a considerable effect on the rate; cis-cyclopentan-2-01 I-phenyl phosphate (11) reacts in base 70 times slower than does tetrahydrofuran-4-01 3-phenyl phosphate (at both 25" and SO'). This substitution of CH2 for 0 would be expected to decrease KZ but to increase k. The net effect on the observed rate (which depends on the product kKz) is a decrease, so that according to this simple analysis the con(t,) (1') stant, analogous to a Brmsted coefficient for this reaction (intramolecular nucleophilic attack on phosphorus and that the dissociation constants of the phosphate and by the alkoxide anion) would be less than one. A hydroxyl groups are ir~dependent,~C the fraction of EH similar explanation can be given for the slower rate of that is present as HE(d) will be 2.3 X IO-l3. Reaction base-catalyzed ring closure of some esters of 3-hydroxyby way of the kinetic equivalent (d) would then require propylphosphonic acid compared with the related 2that this species decompose with a specific rate of 2 X hydroxyethyl phosphate^,^^ as in an intermolecular reacIO6 sec-', so that protonation of the phosphate anion tion the phosphonate may be expected to react faster.73 could speed up attack by the neighboring alkoxide by a Cyclopentanediol thus represents a relatively poor factor of as much as lo5. This seems reasonable in model for ribose, as found previously by Zachau and coview of the reduction in electrostatic repulsion, and it is workers in the case of models of a m i n ~ a c y l - t - R N A . ~ ~thus questionable whether the binding of the phosphate In base, as under all other conditions used in this ingroup of a substrate to a protonated imidazole of vestigation, the cis-methoxy phenyl ester (V) was stable, RNase should be described7' as playing no catalytic role a type of observation that was first made many years in the enzymic reaction; presumably a part of this posa g ~ The . ~rate~ of ~hydrolysis ~ ~ of the cyclic phosphate sible rate increase could be realized by a favorably located general acid catalyst. monoanion, thus k d 3 kc > k , > k b . If kt, were as high as 108sec-l, which is not impossible,eS then k, would lie in the region of the diffusion The base-catalyzed decomposition of the triester (e) controlled limit,63 1010 to 1011 sec-1, which is faster than the probable would constitute a model for the neutral reaction (d). rate of protonation of the dianion by water (about lo9 sec-1 assuming a This compound is not yet available, but a substitute pK, of 13 for the monoanionel). From the above inequality, kc > kt, and since kt, would be expected to exceed the rate of pseudorotation ( k , ) triester, uridine 3 '-dimethyl phosphate, undergoes what of a dianionic intermediate that had both PO- groups in basal posiis apparently base-catalyzed hydrolysis7* with a rate tions,65?68it follows that kc > k , as assumed above. An alternative to constant of possibly 80 M-l sec-l at 37". If the sensithe inequality k , > k b is that the decomposition of the monoanion is highly selective and expels the better leaving group more than 100 times tivity of this reaction to the pK, of the leaving group is faster than methoxyl. This explanation would be more consistent with the high percentage of base-catalyzed exocyclic cleavage noted89 with the analogous phenyl ester. (68) E. L.Muetterties and R. A . Schunn, Quart. Rev. (London), 20, 245 (1966), and references therein. (69) E. A . Dennis and F. H. Westheimer, J. Amer. Chem. Soc., 88, 3431 (1966). (70) D. M. Brown and D. A. Usher, J . Chem. Soc., 6558 (1965). (71) See Table I, footnote g . (72) F. W. Westheimer, personal communication. (73) J. R. Cox, Jr., and 0. B. Ramsay, Chem. Rev., 64, 317 (1964). (74) H . G . Zachau and W. Karau, Ber., 93, 1830 (1960).
Journal oj'the American Chemical Society
1 92:15 1 July
(75) 0.Bailly and J . GaumC, Bull. SOC.Chim. Fr., [SI 3 , 1396 (1936).
(76) Calculations of this sort that wrongly employ macroscopic in place of microscopic dissociation constants can obviously give misleading results. In the present case, the true concentration of HE will be larger than the value calculated by using the macroscopic constants (KI and Kz)and the true rate constant therefore smaller. (77) G. C. K. Roberts, E. A. Dennis, D. H. Meadows, J. S. Cohen, and 0. Jardetzky, Proc. Nar. Acad. Sci. U.S., 62, 1151 (1969). (78) D. M. Brown, D. I. Magrath, and Sir. A. R. Todd, J . Chem. SOC., 3496 (1955).
29, 1970
4709
similar to that of the diester reaction, the rate constant for (e) would be about 105-106 M-l sec-l, and with the pK, of the neighboring hydroxyl probably also about 13-14, this is of the right order for the process d. If the reaction were as shown in (d) and went via a pentacoordinate intermediate, the rate of protonation at 25 O of this initially monoanionic intermediate would probably be about lo6 sec-l by solvated hydrogen ion at pH 4, and 105 sec-I by water63 (assuming a pK, of 9 for These the fully protonated pentaoxypho~phorane).~~~~~ rates could then be less than the rate of decomposition of the monoanionic intermediate.67 Concerted reactions such as (f) can be written for the neutral or acid-catalyzed processes, but since these would involve an “adjacent” type of displacement,6j the preference rulesE0 would require pseudorotation of a pentacoordinate intermediate and it is therefore doubtful whether these could be considered “concerted” in the sense of a four center reaction. The maximum contribution from the neutral pathway to the total rate occurs at pH 4.7 (92.5%); at this pH the terms in H2, H, and OH account for 0.012, 3.7, and 3.875, respectively (see Figure 2). The Reaction Dependent on (H). The contribution of the term in (H) is a maximum at pH 2.8 (63%). At this pH the other terms account for (Hj2, 17x; neutral, 2075; base, 0.01 75,and a bad fit to the experimental points would result if this term in (H) were omitted, as suggested by the reasonably small standard deviation of the rate constant kz (f5 75). By contrast, the acid-catalyzed hydrolysis of nucleoside cyclic phosphatesz5 and dinucleoside phosphates? apparently shows no evidence for a term in the free acid. It is possible that such a term exists, as found here and for dimethyls1 and dibenzyl phosphate,Ezbut in view of the slowness of the reaction of most of these compounds in the region of pH-2-4, where such a term would be most easily seen (being masked by the (H)z term at lower pH) its detection would require careful work at high temperatures. Unlike the neutral and the base terms, the term in (EH)(H) does not necessarily involve reaction via the cyclic phosphate, as none was detected among the hydrolysis products of I in 0.1 M hydrochloric acid (though this term accounts for only about 775 of the reaction under these conditions). However, in view of the stability of the methylated ester V, and the rather small acceleration that would be expected from the hydroxyl group of I if it participated merely as an intramolecular general acid, we consider the existence of such an intermediate probable. The known acid-catalyzed migration of an alkyl phosphoryl group64or of the phosphate group itselfs3 to an adjacent hydroxyl clearly involves a cyclic structure, whether the migration goes by way of a cyclic triester, a pentaoxyphosphorane, or a mixture of both as demonstrated recently for glyc e r o p h ~ s p h a t e . ~Our ~ inability to detect the cyclic diester among the products of acid hydrolysis is the ex(79) As has been pointed out,oo the pKs’s of the basal and apical OH groups would be expected to differ, and also therefore their rates of protonation by water (but not by the solvated proton). (80) F. H. Westheimer, Accounts Chem. Res., 1, 70 (1968). (81) C. A. Bunton, M. M. Mhala, K. G. Oldham, and C. A. Vernon,
J . Chem. Soc., 3293 (1960). (82) J. Kumamoto and F. H. Westheimer, J . Amer. Chem. Soc., 77, 2515 (1955). (83) W. D. Fordham and J. H. Wang, ibid., 89,4197 (1967).
I
I
K Contribution
I
I
I
I
I
I
I
t o Lob,
Figure 2. Per cent contributions to kobsd from the terms in kl, kz, k3,and kn.
pected result of the ring closure now being slower than M-’ the ring opening.5” The value of kz (9.00 X sec-l) refers to the bimolecular reaction of the anion (EH) with hydrogen ion. This corresponds to a firstorder rate constant of 2.5 X sec-’ for the kinetic equivalent (g), which is not very different from the uncatalyzed rate of hydrolysis of uridine 3’-dimethyl phoshate,^^ allowing for the difference in the leaving group.s4 This process could be drawn as being aided
by a molecule of water, acting as a general base.z7 Calculation of the specific rate for the alternative process (h) gives a value of about 4 X I O t 2 sec-’, using an estimate of lo3 for the acid dissociation constant of the conjugate acid of HEH.E6v86However, in view of the uncertainties in assigning pK, values to the phosphate and hydroxyl groups,76 we may not at present exclude (h) on the grounds that it would need to exceed the accepted maximum rate for the separation of products. The Reaction Dependent on (H)z. About 93 of the observed rate of hydrolysis of I in 0.1 M hydrochloric acid is due to the reaction of the conjugate acid of HEH, or its kinetic equivalents. The products of the reaction were phenol and the acyclic monoester, and again the evidence for the existence of the cyclic phosphate as an intermediate is indirect: the stability of the cis-methoxy ester, and the known acid-catalyzed migration of a phosphate group to a neighboring hydroxyl. The migration experiments of Fordham and Wangs3 also suggest that a pentacoordinate intermediate may occur in related reactions in strong acid. The value of kl (0.042 M-’ sec-l) represents the reaction of the free acid plus a proton (HEH)(H); this would require the kinetic equivalent (i) to decompose with a rate constant of 42 sec-’, assuming a value of -3 for pKs as before. The need to use acid stronger than 0.1 M in order to see the dependence of the rate on hydrogen ion start to change from second to first order has been noted pre(84) It is of course possible that the neutral hydrolysis of uridine dimethyl phosphate involves reaction by the equivalent of process h. (85) P. Haake and G. Hurst, J . Amer. Chem. Soc., 88, 2544 (1966). (86) C. A. Vernon, Special Publication No. 8, The Chemical Society, London, 1957, pp 29-30.
Usher, Richardson, Oakenfull
1 Models of Ribonuclease Action
4710
(9 viously with phosphate esters; 2 5 apparently the inductive effect of the ribose or tetrahydrofuran ring is sufficient to lower the pK, of the phosphate group well below In the case of ethylene phosphate, measurements in perchloric acid solutions up to 0.15 M were sufficient to define the pK, as 1.0 (30’; ionic strength O.2),’3!88 whereas for nucleoside cyclic phosphates Abrash, et a1.,25 were unable to detect any departure from the second-order dependence on hydrogen ion up to 0.1 M acid. Interestingly, ethylene cyclic phosphate resembled the nucleoside cyclic phosphates in showing no evidence for a term in (H) at lower acidities,ss and a similar observation has been made for glycerol cyclic pho~phate.*~ Values of kllK1 and k, have also been determined for the cyclopentane phenyl ester I1 at 50’. The observed rate in 0.1 M acid depends almost entirely on the value of k,/Kl,and at this acidity the observed rates are almost identical; Kl for 11 is probably less than for I and thus may compensate for a somewhat smaller kl. This behavior is in contrast to the large reactivity difference between the two esters in base. Elementary Steps of Ribonuclease Action. An attempt has recently been made to estimate the rate constants for some of the proton transfer reactions in one possible mechanism for ribonuclease action (see Scheme 11). 19390
Scheme 11. A Hypothetical Stepwise Proton Transfer Mechanism for Ribonuclease Action’g~~O
1 VI1
-+
formation and breakdown of intermediate
\
CH,
1 -
VIII (87) Sugar phosphates have been known to be stronger acids than monoalkyl phosphates for many years: W. D. Kumler and J. J. Eiler, J . Amer. Chem. SOC.,65,2355 (1943). (88) J. R. Cox, Jr., Ph.D. Thesis, Harvard University, 1959; F. H. Westheimer, Special Publication No. 8, The Chemical Society, London, 1957, pp 1-15. (89) L. Kugel and M. Halmann, J . Amer. Chem. SOC.,89,4125 (1967).
Journal of the American Chemical Society
92:15
If the ratio of the acid dissociation constant of the 2’OH to that of the enzyme base B1 were IO-’ then the rate of formation of the active species VI11 from VI1 (k,) would be about IO6 sec-l, and the fraction of the enzyme substrate complex present as VI11 would be Since the overall reaction for a good substrate (e.g. CpA) is observed7 to proceed at a rate of IO3 to 104 sec-l, this would require that the formation and break down of any pentacoordinate intermediate occur with rate constants of the order of 1Olo to 10” sec-I. l 9 This figure is probably rather high since the difference in the dissociation constant of the 2’-OH and of B1 will be reduced on formation of the enzyme substrate complex. 1 2 If the effect of a diester substrate on the pK, of B1 is similar to that of a monoester i n h i b i t ~ r and , ~ ~ if the pK, of the 2’-OH is somewhat reduced by the proximity of the positively charged active site, it is possible that the required rate of reaction of VI11 may be lowered to about IO8 to IO9 sec-l. Some idea of whether such a large rate would normally be possible for VI11 can be gained from the neutral reaction of the phenyl ester I. It has been shown above that reaction via the kinetic equivalent (d) would require a decomposition rate of about IO6 sec-l at 50°, and irrespective of the actual mechanism of the neutral reaction, the rate of (d) therefore cannot be greater than this value. If the reaction of (d) went by way of an intermediate, present in a low steady state concentration, then since the partitioning of this intermediate would be almost entirely in favor of product formation (phenoxy being a better leaving group than alkoxy) the maximum rate calculated above would refer to the rate of formation of this intermediate. Apart from the different inductive effect of the phenyl group in the model compound, this figure could also represent the upper limit to be expected for the rate of the related reaction of VI11 (in which the phosphate group is not “fully” protonated) and according to this analysis would be insufficient to explain the rate of the enzymic reaction.92 One could conclude that two general acids (presumabIy one histidine and ly~ine-41)9~ may be required to bind the phosphate group of a diester substrate (the upper limit could now be taken as the rate of the process (h), or that the enzyme binds the dinucleoside phosphate in such a way that the conformation of the groups about phosphorus resembles that in a cyclic phosphate. The molecular orbital calculations of Collins4indicate considerable sensitivity of the charge on phosphorus to the orientation of the ester groups, and he has suggested that this factor may account for a good deal of the lability of five-membered cyclic phosphates toward acid or base. It is most interesting, therefore, that the torsion angle around the 3’-0P bond (90) A similar treatment has been given by J. H. Wang (Science, 161, 328 (1968)). (91) D. H. Meadows and 0. Jardetzky, Proc. Nut. Acad. Sci. U . S . , 61,406 (1968). (92) Another kinetic equivalent of the neutral reaction involves protonation of the phenolic oxygen and attack by the alkoxide ion. This process has a maximum specific rate much higher than that of (d), and an analog of this alternative process would simply require the enzyme general acid BH+ (or BIH+) to be in close proximity to the 5 ’ oxygen, The rate of the base-catalyzed reaction of I is a function of the pKa of the leaving group and presumably this would also be true of the neutral reaction so that partial protonation of the 5’-oxygen in VI11 by BH+ or BIH+ could have a large effect on the real rate expected for this reaction.’9,90 (93) The relationship of the cross-linking experiment of Marfey and coworkers ( J . Biol. Chem., 240, 3270 (1965)) to the other lysine modification studies has not yet been satisfactorily resolved. (94) R. L. Collin, J . Amer. Chem. Soc., 88, 3281 (1966).
July 29, 1970
471 1
corresponds to a maximum in reactivity for an “in-line” mechanism, and near the minimum for either of the two “adjacent” mechanism^.^^ The considerable specificity of vmax with respect to the 5’-linked nucleoside of a dinucleoside phosphate substrate’ could be consistent with this variation of the “strain” theory of enzymic catalysisg5 However, there is no requirement for this process to occur by way of individual steps as shown in Scheme 11, and reaction via a “concerted” general base-general acid type of mechanism bypassing VI11 could allow formation of a pentacoordinate intermediate or of products at a faster rate than would obtain if reaction had to proceed via a vanishingly small concentration of a species with high but finite reactivity. Acknowledgments. We thank Dr. G. G. Hammes for the use of his stopped-flow apparatus, Drs. C. F. Wilcox and W. P. Jencks for numerous discussions, and Drs. F. H. Westheimer and S. J. Benkovic for making available copies of their manuscripts prior to publication. Appendix Least Squares Fitting of pH-Rate Profiles. Methods used to fit an assumed theoretical equation to an experimental pH-rate profile vary widely among different investigators; in many cases no explicit mention is made of the actual curve-fitting process, and no estimate is given of the confidence to be placed in the parameters. This situation is in contrast to other fields in which relatively sophisticated techniques are regularly employed. It is therefore hoped that the following details of the curve fitting, when read in conjunction with the paper of Wentworth,47will prove helpful. The equation for kobsd(eq 1) is rearranged to give
For obvious reasons the fit is made with H and kobsd (rather than with pH and log kobsd). If we define a = (k,H2/K1 k2H k3 k4Kw/H)and P = k / ( R H ) , then the partial derivatives of F with respect to H, kobd, k l , k2, k,, k4, and k are
+
+ +
-b-F bkobsci
+
The partial derivatives are calculated for all N experiH)J. mental points, of which the Jth is (kobsd, We estimate the standard deviation of our pH measurements to be a constant 0.01 unit, thus since d(1og H ) = d(ln H)/2.303 = d(H)/2.303H, the variance of H = crH2 = 5.3 X ( H ) 2 . Similarly, the standard deviation of kobsd is about 5 throughout the pH range, thus the Variance Of kobd = b k o b a d 2 = 2.5 x ioF3(kobsd)’. The variance of unit weight was arbitrarily chosen as 10-13,so that
L =
[
’(E)*] +
k 0 b s d ~ [ ~x -5
H 2 5.3 X 10
1010(aE)
The inclusion of weighting is essential, in a typical pHrate profile the value of H alone may vary by a factor of more than lolo, and neglect of the weighting factor will not only invalidate the calculated standard deviations of the parameters, but can result in a curve that even to the eye is a poor fit. If we let the partial derivatives of F with respect to kl, k2, k3, kq, and k , evaluated at the Jth point, be F(1, J ) , F(2, J ) , F(3, J), F(4, J), and F(5, J ) respectively, then the 5 X 5 matrix (B) of the coefficients of the normal equations for N points can be simply generated by the following Fortran statements (without even taking advantage of the symmetry of ( B ) )
DO 1 K = 1,5 DO 1 L = 1,5 B(K,L) = 0. D O l J = l,N B(K,L) = B O W ) 1 CONTINUE
+ F(K,J)*F(L,J)/F(6,J)
where F(6, J ) is the value of L, calculated at the Jth point. The column matrix (C) of the constants from the right-hand side of the normal equations is then calculated
DO 2 K = 1,5 C(K) = 0. DO2J= l,N C(K) = C(K) F(K,J)*FI(J)/F(6,J) 2 CONTINUE
+
-
-I
( 9 5 ) The analogy of the ring strain of a cyclic phosphate with the strain possibly introduced in a substrate by an enzyme has been mentioned by several workers (see some of the reviews in ref 7 above). For a recent discussion of this point see W. P. Jencks, “Catalysis in Chemistry and Enzymology,” McGraw-Hill Book Co., Inc., New York, N. Y., 1969.
where FI(J) is the value of the original function, calculated at theJth point. The ( B ) matrix is then inverted and the correction terms and variances and covariances calculated as in Wentworth’s article. The corrected values of the parameters are then used in a new round of calculations and the whole process repeated until convergence is obtained. The variance ratio ( F ) test can then be applied, if desired, to test the overall goodness of fit. In the present work, the use of pH-rate data down to pH 1.1 gave a very poor definition to the separate values of k, (0.21 with a standard deviation of 0.42) and K1 (1.6 with a standard deviation of 3.3). However, for the reasons outlined in the Results, the ratio kl/Kl is well defined. This is then an example4’ where the variance of a quantity derived from the least squares parameters is reduced greatly by the inclusion of the covariance term. Let g = k,/Kl, then Usher, Richardson, OakenfuN
1 Models of Ribonuclease Action
4712 a fit “by eye.”96 The present example is instructive in that the investigator would have been warned by the large standard deviations of kl and Kl that the data did not adequately define these two constants even if the reason for this had not been recognized. This warning would not necessarily have been apparent with other more intuitive fitting methods.
where uklK,is the covariance of kl and Kl. The three terms of this equation were numerically $0.06491, +0.06992, and -0.13472, so that the value of k l / K l (0.13 1) has a standard deviation of only 8 %. We feel that the use of a general procedure of this sort is much to be preferred over methods that rely on special characteristics of a particular problem or which obtain
(96) This type of rough fit (e.g., with a CRT on-line to a digital computer) is a convenient way of obtaining the initial trial parameters for the least squares program.
Stereochemistry of a Substrate for Pancreatic Ribonuclease. Crystal and Molecular Structure of the Triethylammonium Salt of Uridine 2’,3’-0,0-Cyclophosphorothioate1 W. Saenger and F. Eckstein
Contribution f r o m the Max-Planck-Institut f u r experimentelle Medizin, Abteilung Chemie, 34 Gottingen, Germany. Received December 31, 1970 Abstract: Uridine 2’,3 ’-0,O-cyclophosphorothioate is a substrate for pancreatic ribonuclease. Knowledge of the absolute configuration of this substrate and of the reaction product obtained by enzymatic methanolysis is of interest from a mechanistic point of view. The title compound crystallizes in space group P212121. The structure was solved from three-dimensional X-ray data and refined to an R value of 9.6%. The bicyclic system is “pseudomirror” symmetrical with respect to the 0-P-S plane. The ribose exhibits the unusual O(1’) exo conformation, the bonds C(l ’)-C(2’), C(3’)-C(4’) and C(2’)-0(2‘), C(3’)-O(3‘) being pairwise coplanar. The heterocycle is in anti position with respect to the ribose and almost coplanar with the C(l ’)-O(1 ’) bond. The conformation about the C(4‘)-C(5’) bond, defined as pooand pot, is trans,gauche. The P-S bond seems to have double bond character, the negative charge being located at the free oxygen atom of the phosphorothioate group. The triethylammonium cation is disordered, its symmetry nearly CZv.
R
ecently the uridine 2’,3’-O,O-cyclophosphorothioate anion was synthesized* and one of the diastereomers isolated by fractional crystallization of the triethylammonium salt (Figure 1). This isomer is a substrate for pancreatic ribonuclease with the same K , value as uridine 2’,3’-O,O-~yclophosphate. In the presence of methanol it is converted by the enzyme to uridine 3 ‘-0-(0-methyl)phosphorothioate, which we were able to crystallize as well. Knowledge of the absolute configuration of substrate and reaction product would clarify the stereochemistry of the enzymatic methanolysis and would thus yield valuable information as to the stereochemistry of the transesterification and hydrolysis reaction of ribonuclease. In this report the X-ray structural analysis of the triethylammonium salt of the crystalline isomer of uridine 2’,3’-O,O-cyclophosphorothioatewill be presented, which is interesting not only from a biochemical but also from a structural point of view since nucleoside 2 ’,3 ’-0,O-cyclophosphates have not yet been investigated. (1) Short communication published in A n g e w . Chem. I n f . Ed. Engl., 8, 595 (1969). Differences in some angles and bond distances in this and the present publication are due to the disorder of the triethylammonium cation which had not been accounted for at that time. (2) F. Eckstein and H. Gindl, Chem. Ber., 101, 1670 (1968). (3) F. Eckstein, FEBS (Fed. Eur. Biochem. Soc.) Left., 2 , 85 (1968); 6 values for uridine 2’,3 ’-0,O-cyclophosphorothioate in this publication
should be corrected to 6 = - 68.5 and - 69.5 ppm, respectively. (4) M. R. Harris, D. A . Usher, H. P. Albrecht, G. H. Jones, and J. G. Moffatt, Proc. Nat. Acad. Sci., 63,246 (1969).
Journal of the American Chemical Society 1 92:15 I JuIy 29, 1970
Experimental Section When an ethanolic solution of the triethylammonium salt of uridine 2’,3 ’-0,O-cyclophosphorothioate was allowed to evaporate slowly at 4” stout prismatic crystals formed (mp 204-205’). The orthorhombic space group P212121of these crystals was indicated by the mirror symmetries of the X-ray photographs and the systematic absence of reflections hOO, OkO, 001 when h,k,1, respectively, were odd. The unit cell dimensions werqa = 12,495 + 0.005 A , b = 7.079 i 0.003 A , c = 22.574 i 0.008 A. The observed density of 1.391 =k 0.005 g/cm3 was in good agreement with the calculated value if four formula weights within the unit cell were assumed. We collected 1108 data on a four-circle automatic diffractometer using Zr-filtered Mo radiation and 20 scan mode. In view of the small crystal dimensions of 0.3 X 0.1 X 0.1 mm and the linear absorption coefficient of 2.83 cm-1 the data were corrected for geometrical factors, not for absorption. The positions of the two heavy atoms, P and S, could be determined unambiguously from a sharpened Patterson map. Two successive Fourier syntheses, phased first with these two atoms and then with all but three atoms of the structure, revealed the geometry of the molecule. After two cycles of isotropic and four cycles of anisotropic full-matrix least-squares refinement using the 986 observed data and applying Hughes’ weighting scheme6(Fomin = 4.2), the reliability index, R = ZIIF,( - IFc///ZIFoI, dropped to 9.4%. A difference Fourier synthesis computed at this stage revealed a statistical disorder of the three a-carbon atoms of the triethylammonium ion. In a further cycle of refinement the occupation and thermal parameters of the six “ordered” and “disordered‘’ a-carbon atoms of the ethyl groups were set at 0.5 and isotropic, respectively, and varied. Since the occupation parameters fluctuated around 0.5 but did not shift in a definite manner they were reset to 0.5, the isotropic thermal parameters converted (5) E. W . Hughes, J . Amer. Chem. SOC.,63, 1737 (1941).
