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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 ribonu- clease 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 condi- tions. All buffers used for kinetics were prepared with deionized Im--'' -HO H20 (Millipore). The pH of all aqueous solutions was mea- sured 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.
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