On the Mechanism of Action of Ribonuclease A: Relevance Of

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 10018-10021, September 1996 Chemistry

On the mechanism of action of ribonuclease A: Relevance of enzymatic studies with a p-nitrophenylphosphate ester and a thiophosphate ester RONALD BRESLOW AND WILLiAM H. CHAPMAN, JR. Department of Chemistry, Columbia University, New York, NY 10027 Contributed by Ronald Breslow, June 20, 1996

ABSTRACT It has been reported that His-119 of ribonuclease A plays a major role as an imidazolium ion acid catalyst in the cyclization/cleavage of normal dinucleotides but that it is not needed for the cyclization/cleavage of 3'-uridyl p- [is-12 H;is-12 nitrophenyl phosphate. We see that this is also true for simple buffer catalysis, where imidazole (as in His-12 of the enzyme), 'HIm but not imidazolium ion, plays a significant catalytic role with the nitrophenyl substrate, but both are catalytic for normal 0-1 dinucleotides such as uridyluridine. Rate studies show that the enzyme catalyzes the cyclization of the nitrophenylphos- 2-proton shift HO, phate derivative 47,000,000 times less effectively (kcat/kuncat) I I R than it does uridyladenosine, indicating that '50% of the HiS-1 19 His-I 19 catalytic free energy change is lost with this substrate. This suggests that the nitrophenyl substrate is not correctly bound FIG. 1. Classical mechanism for cyclization-cleavage of RNA by to take full advantage of the catalytic groups of the enzyme ribonuclease A, still favored by some workers. and is thus not a good guide to the mechanism used by normal there was a mechanism for cleavage of 3',5'-uridyluridine nucleotides. The published data on kinetic effects with ribo- (UpU), in which the first step was reversible protonation of the nuclease A of substituting thiophosphate groups for the substrate by imidazolium ion, then cyclization to a phos- phosphate groups of normal substrates has been discussed phorane monoanion catalyzed by Im. Some alternatives have elsewhere, and it was argued that these effects are suggestive been suggested (10, 11). Of course, in an enzyme, such a of the classical mechanism for ribonuclease action, not the sequential mechanism would be replaced by a simultaneous novel mechanism we have recently proposed. The details of acid-base process, as in our proposal. Proton inventory studies these rate effects, including stereochemical preferences in the (12) show that indeed the enzyme has two protons "in flight" thiophosphate series, can be invoked as support for our newer in the transition state for catalyzed hydrolysis of the cyclic mechanism. phosphate, as expected for a simultaneous mechanism. Our mechanistic study then inspired us to examine a syn- In the classic mechanism for cleavage of RNA by ribonuclease thetic ribonuclease mimic in which the two Ims were held in A (refs. 1 and 2; ref. 3 and references cited therein), the such a geometry that they favored our new mechanism. We enzyme first catalyzes an ester exchange to convert the 3',5'- (13-15) found that this catalyst was better than one designed phosphodiester link to a 2',3'-cyclic phosphodiester, with to perform the previous standard mechanism. Furthermore, cleavage ofthe chain. Then the enzyme catalyzes the hydrolysis proton inventory studies indicated that our catalyst uses a of the 2',3'-cyclic phosphodiester link to form a 3'- simultaneous two-proton shift mechanism (16), just as the monophosphate group. The three catalytic groups active in the enzyme does. This geometric evidence with a catalyst that enzyme are the imidazole (Im) rings of His-12 and His-119 and actually uses a simultaneous two-proton mechanism, as the the ammonium group of Lys-41. enzyme does, is of course much stronger than is evidence by In the earliest proposed mechanism (Fig. 1), the basic Im analogy with a sequential two-proton mechanism catalyzed group of His-12 assists the attack of the 2'-OH group of the simply by buffer. substrate, while the acidic imidazolium ion of His-119 assists Further evidence for our mechanism comes from data on the departure of the leaving group. This is the mechanism ribonuclease itself (1-3), which indicates that anionic sub- presented in most textbooks. However, we have proposed an strates bind in such a way as to form hydrogen bonds between important modification. In our mechanism (Fig. 2) (4, 5), the His-12 and the 2'-OH group, as both mechanisms suggest, but His-12 has the same function as before, but the His-119 also between the ImH+ of His-119 and the anionic phosphate simultaneously protonates a phosphate oxyanion group, form- oxygen of the substrate, as our mechanism suggests. Computer ing an intermediate phosphorane monoanion. Only later does calculations are also in line with our proposals. Karplus and this intermediate lose the nucleoside group to form the cyclic coworkers (17-21) have done computer simulations on both phosphate product. Presumably with either mechanism the the classical mechanism and our new mechanism, and appar- subsequent hydrolysis of this cyclic phosphate follows the same ently both are possible from their treatment. Wladkowski has pathway in reverse, with a water molecule substituting for the done a related computer study (22) and favors a mechanism hydroxyl group of the now departed nucleoside. like ours in which protonation occurs first on the phosphate Our argument for the new mechanism was based on several oxyanion, not on the leaving group. pieces of evidence. The earliest was our finding (4-9) that Two papers have recently been published favoring the classical mechanism over our newer mechanism. In one study The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviations: Im, imidazole; UpU, uridyluridine; UpA, uridylad- accordance with 18 U.S.C. §1734 solely to indicate this fact. enosine. 10018 Downloaded by guest on September 24, 2021 Chemistry: Breslow and Chapman Proc. Natl. Acad. Sci. USA 93 (1996) 10019 MATERIALS AND METHODS CH2 t Base Triethylammonium p-nitrophenyl-2',5'-ditetrahydropyranylu- ~~Base ~His-12 ridine-3'-phosphate (bis-THP-1) was synthesized from 3'-O- benzoyluridine (25) by the method reported by Davis et al. (26). The product was purified by ion-exchange flash chroma- I! tography [Toyopearl DEAE-650M, 65 ,tm (Tosohaas, Mont-

HO-P-OI gomeryville, PA), equilibrated with 100 mM NH4OAc] with -4 100 mM triethylammonium acetate as the eluent. Lyophilized 2-proton Im R ° bis-THP-1 was found to be a white solid that underwent no shift I NH3+ His- 119 decomposition in 5 months when stored at -20°C. Lys-41 Aqueous solutions of structure 1 for kinetic studies were produced by treatment of bis-THP-1 (1 ml, 10 mM) with water-swollen Amberlite-15 (Sigma) strongly acidic ion- CH2 exchange resin (25-30 mg) for 1 h at room temperature. The ABase aqueous solution was separated from the resin and stored at His-12 0°C. The identity of structure 1 was further confirmed by 1H NMR (400 MHz, D20, 10 mM): 8.13 (d, J = 8.4 Hz, 2 H, Ar); 0\ /0 ;;y Im 7.72 (d, J = 8.1 Hz, 1 H, C,H); 7.24 (d,J = 8.4 Hz, 2 H, Ar), 5.81 (d, J = 6 Hz, 1 H, H1i), 5.74 (d, J = 8.1 Hz, 1 H, C5H), -4- ca. 4.7 (obscured by HDO peak, H3), 4.30 (dd, Ja = Jb = 6 Hz, 2-proton 1 H, J = Hz, 1 H, H4 J = Hz, K H2 ), 4.16 (d, 1.5 ), 3.72 (d, 13 Im II ImH+ R NH3+ shift 1 H, H5'), 3.63 (dd, Ja = 13 Hz, Jb = 1.5 Hz, H5s). All other I His-119 Lvs-41 significant peaks in the spectrum (3.8, 3.4, 1.65, and 1.2-1.4 His-ll9 D}y ;3-- a ppm) were assigned to hydrated dihydropyran (the spent protecting group) by comparison to an authentic sample. After CH2 sitting at room temperature for 6 days, structure 1 was found Base to decompose to nitrophenol and a complex mixture of uridine His- 12 derivatives. Uridine-2',3'-cyclic phosphate (10 mM in D20) was found to produce a mixture with the same spectral features O\ o +HIm when protonated with Amberlite-15 under the same conditions. All buffers used for kinetics were prepared with deionized Im--'' -HO H20 (Millipore). The pH of all aqueous solutions was measured with an Orion (Cambridge, MA) combination electrode R His-i 19 (Ag/AgCl reference) and an Orion 701A digital pH meter at room temperature. Hepes buffer (20 mM; Sigma, ultragrade, FIG. 2. Mechanism we have proposed for the action of ribonuclease >99.5% pure) was titrated to pH 8.35 (90% deprotonation) A, in which the first function of the Im ion of His-119 is to protonate with NaOH (1 M; Fisher, A.C.S. grade). For the Im-catalyzed the phosphate anionic oxygen, leading to a phosphorane monoanion experiments, stock solutions of Im, ImHCl (0.05 M and 2.5 M; intermediate. Sigma) and NaCl (2.5 M; Fisher, A.C.S. grade) were mixed, and the volume was to 1 ml with The concentration of ribonuclease A and various of its mutants were examined brought H20. (23), Im was held constant M or 10 and the concentration of as for the of (0.5 mM) catalysts cyclization/cleavage 3'-uridyladenosine ImHCl was varied (from 4.4 mM to 17.6 mM and from 0.22 M to a normal and also for the (UpA), substrate, cyclization- 0.88 M). The total salt concentration (1.1 M) was maintainedwith cleavage of the p-nitrophenylphosphate ester (1 in Scheme I) NaCl. of 3'-uridylic acid. For the kinetic measurements, structure 1 (10 ,ul of a 10 mM solution) was added to 1 ml of the appropriate buffer, and the solution was mixed as quickly as possible with a Pasteur pipet (final concentration of structure 1, 0.1 mM). The rate of formation of nitrophenylate from structure 1 was measured at 0 400 nm with a Cary-Varian (Palo Alto, CA; model le) dual-beam absorbance spectrometer, while the temperature of the cuvets was maintained at 25°C with a thermostated cuvet B: holder. In all cases, the reported rate constants were calculated > NO2 by the method of initial slopes (<5% conversion, 20 points, r2 > 0.999) with extinction coefficients of nitrophenylate (400 nm, 4 or 5 points, r2 > 0.99), which were measured under identical conditions. The rate of cyclization-hydrolysis of 3',5'-UpU in 20 mM HEPES buffer at pH = 8.35 was measured at four different Scheme I temperatures (91°C, 81°C, 71°C, and 41°C) as has been described (27). The kinetic methods employed were identical to those reported by Breslow and Xu (5). In the other paper (24), some literature data on thiophosphate substrates for ribonuclease were examined in light of the two RESULTS AND DISCUSSION alternative mechanisms. In this paper we will examine the strength of these arguments. We conclude that the existing The Cyclization of Nitrophenylphosphate Ester 1. Thomp- data do not exclude our mechanism, and indeed they furnish son and Raines (23) have examined the enzymatic hydrolysis some additional support for it. of structure 1 and of 3',5'-UpA by native ribonuclease A and Downloaded by guest on September 24, 2021 10020 Chemistry: Breslow and Chapman Proc. Natl. Acad. Sci. USA 93 (1996)

also by mutants in which His-12 or His-119 are replaced by Ala. -13.5 As expected, catalysis of the cyclization of the normal UpA substrate was greatly decreased when either His-12 or His-119 -14.0 - was replaced, but the situation was different with substrate 1. Here replacement of His-12 again led to great loss of activity, -14.5 - but replacing the Im of His-119 caused no rate problem for -15.0 - substrate 1. This indicates that the acid catalytic group of the enzyme is not required with this substrate. -15.5 Thompson and Raines suggested that a p-nitrophenoxide - 16.0 ion is such a good leaving group that it does not require : protonation by an ImH+. However, they pointed out that - -16.5 apparently substrate 1 is also not being protonated on the phosphate oxyanion during catalysis, or loss of His-119 would -17.0 have been a problem. This then raised the question of whether -17.5 such oxyanion protonation, the central feature of our mechanism, occurs with normal substrates. -18.0 We have now examined the chemical reactivity of substrate 1 with buffers as catalysts for the cyclization reaction. Related -18.5 studies have been done previously (26, 28). The compound -19.0 behaves quite differently from a normal substrate such as 2.60 2.70 2.80 2.90 3.00 3.1 0 3.20 3.30 UpU. For example, with [1m] held constant at 0.5 M, we see an increase in the rate of cyclization of UpU as [ImH+] is 1/T (1/K) X 10 increased, while ionic strength is held constant with NaCl (9). FIG. 4. Natural logarithm of the pseudo-first-order rate constant However, with constant [Im] and constant ionic strength, the (s-1) for cyclization/cleavage of 3',5'-UpU in 20 mM Hepes buffer rate of cyclization of structure 1 decreases as [ImH+] is (pH 8.35) at 91°C, 81°C, 71°C, and 41°C, determined by methodology increased (Fig. 3). This rate decrease essentially parallels the that has been described (5, 27). decrease for structure 1 resulting from the lowering of pH, measured with buffers at 2% of these concentrations. Thus ture 1 is -47,000,000-fold (74 x 63,000) less than for normal UpU cyclization is catalyzed by both Im and ImH+, as we have dinucleotide substrates. There is evidence that desolvation of reported earlier, but the cyclization of structure 1 is catalyzed the substrate contributes to the rate of enzymatic hydrolysis of by Im and shows no catalysis by ImH+. This parallels the UpA (29) but not of structure 1, so the rate of the chemical observations with the enzyme. process for UpA is even larger relative to that for structure 1. More importantly, the catalyzed cyclization of structure 1 by Since kcat/kuncat with the best substrates is 1012 for the the enzyme shows a huge loss of catalytic acceleration. We find enzyme, this means that more than half of the normal SAG' for that the cyclization of structure 1 occurs 6.3 x 105 times faster enzymatic catalysis is lost with structure 1. Thompson and than that of UpU with 20 mM Hepes buffer at pH 8.35 and Raines saw that the ImH+ of an enzyme histidine was not 25°C. Under these conditions, kcieavage for structure 1 is 1.52 ± needed for the hydrolysis of structure 1, just as we see it is not 0.06 X 10-3 S-1, while that for UpU is 2.4 x 10-9 s-1 by needed in the buffer-catalyzed reaction. However, with such a extrapolation from four higher temperatures (Fig. 4). How- huge loss of catalytic acceleration, it seems likely that structure ever, the enzyme-catalyzed cyclization of structure 1 has a kcat 1 is not correctly bound to the enzyme, and is thus not a good that is 74 times lower than that for UpA (23). Thus the catalytic guide to the mechanism for normal substrates. Our proposed acceleration kcat/kuncat for the cyclization/cleavage of struc- simultaneous bifunctional mechanism for the enzyme (Fig. 2), involving a phosphorane monoanion intermediate, remains a 2.00 real possibility for the catalyzed reactions ofnormal substrates. 0 Studies with Thiophosphate Analogs ofUpA. Herschlag (24) 1.75 _ has cited prior work by Burgers and Eckstein (ref. 30; for an 0 0 1.50 _ 0 0 0 * nc 1.25 U _ 1.00 _ * U a v 0.75 _ E v v 0.50 _ V V v 0.25 _

0.00 1 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 [ImH ]/[Im] FIG. 3. Cyclization/cleavage of structure 1 by Im buffers. *, [Im] held constant at 0.5 M. v, [Im] held constant at 0.05M. *, The difference between these two curves. Total salt concentration was held 29,Rp 3, Sp constant at 1.1 M with NaCl. Scheme II Downloaded by guest on September 24, 2021 Chemistry: Breslow and Chapman Proc. Natl. Acad. Sci. USA 93 (1996) 10021 earlier use of the thio effect in an enzyme mechanism study, 2. Blackburn, P. & Moore, S. (1982) in The Enzymes, ed. Boyer, see ref. 31) to argue that the known effect of sulfur substitution P. D. (Academic, New York), 3rd Ed., Vol. 15, 317-433. on the rates of enzymatic cyclization of the Rp (2 in Scheme 3. Campbell, C. L. & Petsko, G. A. (1987) Biochemistry 26, 8579- II) and Sp (3 in Scheme II) isomers of Up(S)A are evidence 8584. 4. Breslow, R. (1991) Acc. Chem. Res. 24, 317-324. against our mechanism. His principal arguments deal with 5. Breslow, R. & Xu, R. (1993) J. Am. Chem. Soc. 115,10705-10713. trying to exclude prior protonation of the phosphate in the 6. Breslow, R., Anslyn, E. & Huang, D.-L. (1991) Tetrahedron 47, enzymatic process, but this has never been a serious possibility. 2365-2376. Our mechanism involves a simultaneous two-proton process, 7. Breslow, R. & Xu, R. (1993) Proc. Natl. Acad. Sci. USA 90, consistent with the known proton inventory evidence. How- 1201-1207. ever, he then proposes that the arguments can be extended to 8. Anslyn, E. & Breslow, R. (1989) J. Am. Chem. Soc. 111, 4473- the simultaneous mechanism as well. 4482. He describes a number of caveats with respect to his 9. Breslow, R., Dong, S. D., Webb, Y. & Xu, R. (1996) J. Am. Chem. Soc. 118, 6588-6600. arguments, but there are some other points to be considered. 10. Kirby, A. J. & Marriott, R. E. (1995) J. Am. Chem. Soc. 117, First of all, the Rp isomer has kcat only 2.4-fold less than that 833-834. for the oxygen analog substrate, while kcat for the Sp isomer is 11. Perrin, C. L. (1995) J. Org. Chem. 60, 1239-1243. 70-fold smaller than that for normal UpA (30). The geometry 12. Matta, M. S. & Vo, D. T. (1986) J. Am. Chem. Soc. 108, 5316- of substrate binding into ribonuclease A is such that protona- 5318. tion of the Rp isomer 2 should occur on oxygen, while 13. Anslyn, E. & Breslow, R. (1989) J. Am. Chem. Soc. 111, 5972- protonation of the Sp isomer 3 by His-119 would occur on 5973. sulfur. Since protonation on oxygen should be easier than on 14. Desper, J. & Breslow, R. (1989) J. Am. Chem. Soc. 116, 12081- sulfur, this argues for the protonation process of our mecha- 12082. 15. Breslow, R. & Schmuck, C. (1996) J. Am. Chem. Soc. 118, nism. Secondly, the argument that the kinetic effects of sulfur 6601-6605. substitution are too small to accommodate our process (24) are 16. Anslyn, E. & Breslow, R. (1989) J. Am. Chem. Soc. 111, 8931- weakened by the possibility that the chemical step may not be 8932. the rate-limiting process. Raines and coworkers (29) have seen 17. Haydock, K., Lim, C., Brunger, A. T. & Karplus, M. (1990)J. Am. important solvent effects with wild-type enzyme and normal Chem. Soc. 112, 3826-3831. UpA, indicating that the reaction rate is partly controlled by 18. Lim, C. & Karplus, M. (1990) J. Am. Chem. Soc. 112, 5872-5873. desolvation. This means that the intrinsic S/0 element effect 19. Dejaegere, A., Lim, C. & Karplus, M. (1991) J. Am. Chem. Soc. on the rate of the chemical step may be even larger than the 113, 4353-4355. 20. Dejaegere, A. & Karplus, M. (1993) J. Am. Chem. Soc. 115, gross rate comparisons indicate. 5316-5317. 21. Dejaegere, A., Liang, X. & Karplus, M. (1994) J. Chem. Soc. CONCLUSIONS Faraday Trans. 90, 1763-1770. 22. Wladkowski, B., Krauss, M. & Stevens, W. (1995) J. Am. Chem. The enzymatic data with abnormal substrates such as the Soc. 117, 10537-10545. p-nitrophenylphosphate ester 1 or the thiophosphate diesters 23. Thompson, J. E. & Raines, R. T. (1994) J. Am. Chem. Soc. 116, 2 and 3 do not exclude our novel mechanism for the action of 5467-5468. ribonuclease A on its normal substrates, and, indeed, they 24. Herschlag, D. (1994) J. Am. Chem. Soc. 116, 11631-11635. furnish some arguments in favor of that mechanism. 25. Wagner, D., Verheyden, J. P. H. & Moffatt, J. G. (1974) J. Org. However, Chem. 39, 24-30. only further work will establish definitive evidence for or 26. Davis, A. M., Hall, A. D. & Williams, A. (1988)J. Am. Chem. Soc. against our proposal. 110, 5105-5108. 27. Breslow, R. & Chapman, W. H., Jr. (1995)J. Am. Chem. Soc. 117, We thank Ronald Raines for helpful comments and for making 5462-5469. some of his data available before publication. Support of our work by 28. Davis, A. M., Regan, A. C. & Williams, A. (1988) Biochemistry the National Institutes of Health, including a National Institutes of 27, 9042-9047. Health Postdoctoral Fellowship to W.H.C., is gratefully acknowl- 29. Thompson, J. E., Kutateladze, T. G., Shister, M. C., Venegas, edged. F. D., Messmore, J. M. & Raines, R. T. (1995) Bioorg. Chem. 23, 471-481. 1. Richards, F. M. & Wyckoff, H. W. (1971) in The Enzymes, ed. 30. Burgers, M. J. & Eckstein, F. (1979) Biochemistry 18, 592-596. Boyer, P. D. (Academic, New York), 3rd Ed., Vol. 4, 647-806. 31. Breslow, R. & Katz, I. (1968) J. Am. Chem. Soc. 90, 7376-7377. Downloaded by guest on September 24, 2021