Biochemistry 0 Copyright 1983 by the American Chemical Society
Volume 22, Number 13
June 21, 1983
Accelerated Publications Exceptional Characteristics of Amino Proton Exchange in Guanosine Compoundst Bruce McConnell,* Douglas J. Rice, and Francis-Dean A. Uchima
ABSTRACI:
Amino ‘H NMR line width as a measure of amino proton exchange in guanosine compounds is completely unaffected by the addition of ca. 1 M tris(hydroxymethy1)aminomethane, imidazole, 2-(N-morpholino)ethanesulfonic acid, glycine, or cacodylate, all shown to be effective buffer catalysts in adenosine and cytidine proton exchange. Line broadening, seen only with phosphate and acetate, is established by intermolecular interactions, as well as by amino to
water proton exchange. This absence of buffer catalysis of exchange is accounted for by the relatively small implied effect of G(N-7) protonation on amino acidity, based on similar observations with 7-methylguanosine as a model for endocyclic protonation. The requirement for diffusion-controlled proton transfer in buffer catalysis is achieved by nucleobase protonation in adenine and cytosine, but not in guanine.
B u f f e r catalysis of nucleobase hydrogen exchange has been demonstrated for the amino group of adenosine compounds (McConnell & Seawell, 1972; McConnell, 1974,1978a; Cross et al., 1975), the amino group of cytosine compounds, and the imino (N-1) group of guanylic acid (McConnell, 1978a). The mechanism of buffer catalysis for the amino protons of adenine and cytosine was shown to involve the exclusive roles of buffer conjugate base as acceptor and the endocyclic protonated nucleobase as amino proton donor; the neutral, unprotonated nucleobase does not contribute to observed buffer-catalyzed exchange and gives up its amino protons only to hydroxyl ion at measurable rates (McConnell, 1974, 1978a). More recent N M R studies have been made (B. McConnell and D. Politowski, unpublished results) that lend support to this mechanism through a comparison of buffer catalysis in amino proton exchange of adenosine, 1-methyladenosine, cytidine, and 3methylcytidine. As models for endocyclic protonation, the endocyclic methylated nucleobases are about 1O4 more acidic than the unmodified (and unprotonated) compounds at their amino sites, and their amino proton exchange conforms to a relationship between second-order buffer rate constant and buffer dissociation that is expected for diffusion-limited encounters. Thus, buffer-catalyzed exchange in adenine and cytosine is a function of both buffer pK and nucleobase pK, since endocyclic protonation is required to provide a sufficient
increase in amino acidity for the observation of proton transfer to a solvent acceptor. This kinetic route was postulated as a mechanism for amino hydrogen exchange in double helical polynucleotides as well (Teitlebaum & Englander, 1975). Earlier studies showing phosphate-induced broadening of the amino ‘H NMR signal in guanylic acid led to the expectation of a similar mechanism, which would provide predictable buffer-induced exchange rates based on the magnitude of the guanine (N-7) pK value (McConnell, 1978b). However, the present study shows that phosphate catalysis is an exception. Other buffers, shown to be effective catalysts of amino proton exchange in adenosine and cytosine, are completely ineffective as catalysts in guanylic acid. An explanation for this lack of buffer catalysis is derived from studies with 7methylguanosine whose amino proton exchange is equally insensitive to the addition of buffer (with the exception of phosphate). The 50-fold increase in amino acidity that occurs with N-7 methylation of guanine is insufficient to provide diffusion-controlled rates for hydroxyl-catalyzed exchange and is much smaller than the 104-foldacidity increase observed with methylation of adenine (N-1) or cytosine (N-3). Thus, guanine amino exchange represents a special case, confirming the mechanism of buffer-catalyzed amino proton exchange in adenosine and cytosine that involves the exclusive kinetic role of the protonated nucleobase at neutral to acidic pH values.
From the Department of Biochemistry and Biophysics, John A. Bums School of Medicine, University of Hawaii, Honolulu, Hawaii 96822. Received March 15, 1983. This work was funded in part by National Science Foundation Grant PCM 77-201 54.
Materials and Methods The guanosine compound guanosine cyclic 2’,3’-monophosphate (Sigma) was used as supplied, and 7-methylguanosine was prepared from guanosine by the method of
0006-2960/83/0422-3033$01 .50/0 0 - 1983 American Chemical Societv
3034 B I O C H E M I S T R Y
ACCELERATED PUBLICATIONS
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PH pH dependence of amino 'H NMR line width (WW)and catalytic line broadening (W,) in aqueous 2',3'-cGMP solutions. Data were collected at 27 2 OC at 300 or 360 MHz for 0.03 0.01 M nucleotide without addition (e)and with 0.5 M phosphate (H), 0.75 M phosphate (A), 0.5 M Tris (V), 1.0 M imidazole (V), 0.8 M cacodylate (X), 1.0 M MES ( O ) , and 1.0 M glycine (A). Data obtained at 100 MHz are included for 0.1 M nucleotide (0). Data of (A) were obtained by subtraction of Wow from the line width in the presence of buffer. FIGURE1:
*
*
Jones & Robins (1963). Sodium phosphate, sodium acetate (Baker), sodium cacodylate, tris(hydroxymethy1)aminomethane (Tris), imidazole, glycine (Sigma), and 24Nmorpho1ino)ethanesulfonic acid (MES) (Research Organics, Inc.) were used as supplied in the highest purity available. All solutions contained 0.0014.002 M Na,EDTA (Sigma) and were pH adjusted with a Beckman Model 4500 digital pH meter and an Ingold combined electrode designed for N M R tubes. Collection of N M R spectra involved the use of the Varian high-resolution 100-MHz spectrometer and the Nicolet 300MHz spectrometer,' both of the Department of Chemistry, University of Hawaii. Spectra from the latter instrument were obtained by Redfield Fourier-transform (FT)pulse acquisition (Redfield, 1978). Earlier spectra were obtained from the Bruker HXS-360 N M R spectrometer of the Stanford Magnetic Resonance Laboratory, Stanford University.z The majority of line width measurements were verified on the Nicolet 1280 computer with Lorentzian line-fitting functions iterated to minimum statistical variance from the digitized data in the spectral region of the amino 'H N M R resonances. Results In Figure 1 the pH dependence of line width at 27 OC of the amino 'H N M R resonance is shown for 2',3'-cGMP alone and in the presence of various buffers previously shown to be effective exchange catalysts of amino proton exchange in adenosine and cytosine compounds (McConnell, 1974, 1978a; McConnell and Politowski, unpublished results). With the I The Nicolet 300-MHz spectrometer was acquired from funds awarded by the National Science Foundation, NSF Grant CHE 8100240. The Bruker HXS-360 NMR spectrometer, a national facility at Stanford University, was supported by NSF Grant GR 23633 and NIH Grant RR 007 1 1.
*
pH dependence of guanine amino 'H NMR line width at 4 OC. Observed line widths, Wobd,were obtained at 300 MHz from solutions containing 0.05 M 2',3'-cGMP, 0.001 M EDTA, and the following additions: 1.O M NaC104 (0),0.5 M sodium phosphate ( O ) , and 1.0 M imidazole (V). Angled arrows from three phosphate points indicate precipitation at 4 OC in the NMR tube. pH measurements and adjustmentswere made at 25 O C . The dotted line shows the pH dependence of Wow at 27 h 2 O C for comparison. FIGURE2:
exception of phosphate, none of the buffers produce line broadening even at 1 M concentration. This is most apparent in Figure IA, which shows no specific buffer broadening with Tris, MES, imidazole, glycine, or cacodylate. The phosphate-induced broadening is concentration dependent only in a narrow pH range between pH 4.5 and pH 6.5. Above pH 7, phosphate produces a sharpening of the amino 'H N M R resonance, which may be related to intermolecular association, as evidenced by precipitation at lower temperatures. A pH profile obtained at 4 OC is shown in Figure 2 for data collected in the presence of 0.5 M phosphate and 1 M imidazole and includes a dashed curve representing the 27 OC data for comparison. Precipitation in the presence of phosphate is observed in both line-broadening pH limbs of Figure 2 (pH 7) and is associated with zero or negative effects on line width, as opposed to the significant broadening between these pH values. The large difference in line width collected at both temperatures above pH 7 (without buffer) reflects the catalytic role of OH- in amino proton exchange between the water site and the neutral (unprotonated) guanine nucleobase. The concentration of OH- is lowered through the effect of decreasing temperature, which decreases K, from (at room temperature) to (at 0 "C). This suppression of OH- catalysis allowed for the observation of imidazole catalysis of amino proton exchange in adenine (McConnell, 1974), but not for guanine, as shown here. The possibility of line-broadening suppression between pH 4.5 and pH 6.5 in response to intermolecular association in the 2',3'-cGMP-imidazole mixture is ruled out by measurements of constant amino 'H N M R line width in solutions of widely varying concentrations of 2',3'-cGMP (5-500 mM, not shown). To examine further the relative contributions of exchange and intermolecular association on amino 'H NMR line width, measurements were taken at several temperatures between pH 5 and pH 6 for 2',3'-cGMP without buffer and in the presence of phosphate and imidazole (Figure 3). Imidazole has no effect whatever, even at 65 OC, where -NHz to H20exchange in the absence of buffer is clearly the chief broadening factor. However, phosphate broadening exhibits a strong, positive temperature dependence, much greater than that in the ab-
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VOL. 22, N O . 1 3 , 1983
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Table I: Comparison of Amino Proton Exchange Catalysis in Adenosine, Cytidine, and Guanosine Compounds
hydroxyl phosphate imidazole Tris H30+
15.8 6.8 7.0 8.2
11 5.9 6.1 6.9 -6.3
7.6 nc n n
11 6.3 6.4 7.5 2.8
11 6.3 (6.0) 6.9 kN and kNH+and the essential exchange irreversibility of kN and k"+ reactions, the observed increase in rate with the addition of buffer is
?rw~ = kA (S-l)
= PNkN
+ PNH+k"+
(1)
where P is the mole fraction, kN and kNH+are pH-dependent first-order rate constants, and WAis the observed increase in amino 'H N M R line width. In the case of adenine and cytosine, PNkN is negligible, owing to the observation that fitted values of k"+ at low pH are large and account for all of the observed broadening at all pH values (and where PN = 1) (McConnell, 1974). In this case
where aH+is hydrogen ion activity, KN is the acid dissociation constant of the nucleobase site, and kB is the fitted secondorder rate constant for proton transfer from NH+ to the buffer conjugate base, B, of the acid-base pair, BH+ * B + H+. "Eigen" plots of kB,Le., log kBvs. buffer pK (pKB),are linear with positive unit slope until PKB IpKD, where pKD is a measure of the amino pK of NH+ (McConnell and Politowski, unpublished results). This conformance indicates diffusioncontrolled rates for the formation of the donor-acceptor encounter complex (Eigen, 1964; Crooks, 1975). The implication is that no buffer catalysis would be seen, were it not for the transformation of the nucleobase amino from a poor proton donor to a "normal" acid as a result of endocyclic protonation. Accordingly, in a nucleobase system associated with significantly lower inductive effects of nucleobase protonation, one would expect to see no buffer catalysis under the premise that PNkN
