General acid-base catalysis in nucleobase amino proton exchange: cytidine

A useful property of DMSO solvent has been exploited to reveal a new catalytic route for cytidine amino proton exchange, relevant to exchange in the macromolecular state, but hidden in aqueous solution. Additional exchange mechanisms in aqueous monomeric cytidine (and adenosine) are obscured by the formation of a fast-exchanging endocyclic-protonated intermediate, which dominates the kinetics. Endocyclic nucleobase protonation could be circumvented in the presence of buffer conjugate acid by the use of DMSO/water solvent, permitting the first unequivocal observation buffer acid-catalyzed exchange from the neutral, unprotonated nucleobase, i.e., general acid catalysis. Because buffer ionization is greatly reduced in DMSO through anion desolvation, nucleobase protonation is suppressed in the presence of buffer acid. Evidence is presented to describe this catalytic route as one involving hydrogen bond formation between the buffer acid and the endocyclic protonation site, C(N-3). Since this same configuration is found in Watson-Crick hydrogen bonding, experiments are presented to demonstrate faster cytidine amino proton exchange with the formation of the G-C base pair in DMSO. The importance of this mechanism in past aqueous monomer studies and in the interpretation of macromolecular (DNA) hydrogen exchange is discussed.     

The significance of the aprotic solvent in monomer studies related to the primary subunit environment in the macromolecule

An advantage of aprotic polar solvent systems in the study of monomer interactions relevant to the macromolecular state is demonstrated with the measurement of nucleoside amino proton exchange rates in DMSO/water mixtures. The DMSO/water solvent provides the first unequivocal observation of general acid catalysis of nucleic acid amino proton exchange, which is undetectable in aqueous solution due to the formation of the endocyclic protonated nucleobase. Suppression of nucleobase protonation in the presence of buffer acid is a consequence of anion desolvation in the aprotic solvent. The detected route of general acid catalysis is demonstrated as a consequence of Watson-Crick H-bonding, leading to the implication that amino chemistry is modulated in the helical state to decrease amino proton lifetime in the closed macromolecular context of conformational information obtained by hydrogen exchange methods. This useful property of the aprotic solvent can be extended to monomeric studies pertaining to specific local site interactions affecting the function and conformation of proteins and nucleic acids.     

Exceptional characteristics of amino proton exchange in guanosine compounds

Amino 1H NMR line width as a measure of amino proton exchange in guanosine compounds is completely unaffected by the addition of ca. 1 M tris(hydroxymethyl)-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.     

Buffer catalysis of amino proton exchange in compounds of adenosine, cytidine and their endocyclic N-methylated derivatives

The use of buffer catalysts having a wide range of pK (dissociation) values (4-12) provides the first estimates of two generally useful empirical parameters of amino proton exchange in compounds of adenine and cytosine. These are a nucleobase amino group dissociation constant (pKD) and the 'encounter frequency' for proton transfer (kD), which can be used to predict amino proton exchange rates. Values of amino pKD fall in the range 8.6-9.4 for the unsubstituted nucleobases and their endocyclic N-methylated derivatives. Similar values of kD are obtained for all nucleobases (1 X 10(8) M-1 s-1). These constants were obtained from a statistical fit of second-order catalytic rate constants for amino proton exchange, measured by amino 1H-NMR lineshape at varying field frequencies (100, 300 and 360 MHz). These results confirm the requirement for buffer conjugate base formation and nucleobase protonation, but point to a different mechanism of exchange at low pH; most probably direct amino protonation for adenine, but not for cytosine compounds. Anionic buffer conjugate bases (phosphate and acetate) show a greater catalytic effect than neutral (nitrogen) bases, especially with cytosine compounds. The use of high concentrations of sodium perchlorate to sharpen amino 1H resonances of 1-methyladenosine is examined, with respect to chemical and rotational exchange and NMR line broadening.     

Exchange mechanisms for hydrogen bonding protons of cytidylic and guanylic acids.

The pH dependence of buffer catalysis of exchange of the C-4 amino protons of cyclic cytosine 2',3'-monophosphate (cCMP) and the N-1 proton of cyclic guanosine 2',3'-monophosphate (cGMP) conforms to an exchange mechanism, in which protonation of the nucleobases at C(N-3) AND G(N-7) establishes the important intermediates at neutral to acidic pH. Rate constants for transfer of the G(N-1) proton to H2O, OH-, phosphate, acetate, chloracetate, lactate, and cytosine (N-3) were obtained from 1H nuclear magnetic resonance line width measurements at 360 MHz and were used to estimate the pK or acidity of the exchange site in both the protonated and unprotonated nucleobase. These estimates reveal an increase in acidity of the G(N-1) site corresponding to 2 to 3 pK units as the G(N-7) site is protonated: At neutral pH the G(N-1) site of the protonated purine would be ionized (pK = 6.3). Determinations of phosphate, imidazole, and methylimidazole rate constants for transfer of the amino protons of cCMP provide a more approximate estimate of pK = 7 to 9 for the amino of the protonated pyrimidine. A comparison of the intrinsic amino acidity in the neutral and protonated cytosine is vitiated by the observation that OH- catalyzed exchange in the neutral base is not diffusion limited. This leads to the conclusion that protonation of the nucleobase effects a qualitative increase in the ability of the amino protons to form hydrogen bonds: from very poor in the neutral base to "normal" in the conjugate acid.     

Proton-transfer kinetics of photoexcited 7-hydroxy-1-naphthalenesulphonic acid in aqueous DMSO solutions.

The monoanion of 7-hydroxy-1-naphthalenesulphonic acid (HNS) undergoes pseudo-first-order dissociation and its conjugate base undergoes second-order protonation in the lowest excited singlet state. The proton transfer kinetics in water containing dimethylsulphoxide (DMSO), up to a mole fraction of about 0.4, have been evaluated as a function of DMSO concentration. At mole fractions above 0.5 of DMSO, proton-transfer does not measurably occur. At mole fractions below 0.5, steady-state and pulsed-source fluorimetries show the rate constant for dissociation to decrease exponentially with increasing mole fraction of DMSO. This is believed to be due to penetration and disruption of the aqueous solvent cage of HNS by DMSO, resulting in impairment of the Grotthus proton-transfer mechanism. The rate of neutralization of the conjugate base by hydrogen ion is found to vary only slightly with solvent composition and depends on the bulk dielectric properties of the solvent.     

Rapid amide proton exchange rates in peptides and proteins measured by solvent quenching and two-dimensional NMR.

In an effort to develop a more versatile quenched hydrogen exchange method for studies of peptide conformation and protein-ligand interactions, the mechanism of amide proton exchange for model peptides in DMSO-D2O mixtures was investigated by NMR methods. As in water, H-D exchange rates in the presence of 90% or 95% DMSO exhibit characteristic acid- and base-catalyzed processes and negligible water catalysis. However, the base-catalyzed rate is suppressed by as much as four orders of magnitude in 95% DMSO. As a result, the pH at which the exchange rate goes through a minimum is shifted up by about two pH units and the minimum exchange rate is approximately 100-fold reduced relative to that in D2O. The solvent-dependent decrease in base-catalyzed exchange rates can be attributed primarily to a large increase in pKa values for the NH group, whereas solvent effects on pKW seem less important. Addition of toluene and cyclohexane resulted in improved proton NMR chemical shift dispersion. The dramatic reduction in exchange rates observed in the solvent mixture at optimal pH makes it possible to apply 2D NMR for NH exchange measurements on peptides under conditions where rates are too rapid for direct NMR analysis. To test this solvent-quenching method, melittin was exchanged in D2O (pH 3.2, 12 degrees C), aliquots were quenched by rapid freezing, lyophilized, and dissolved in quenching buffer (70% DMSO, 25% toluene, 4% D2O, 1% cyclohexane, 75 mM dichloroacetic acid) for NMR analysis. Exchange rates for 21 amide protons were measured by recording 2D NMR spectra on a series of samples quenched at different times. The results are consistent with a monomeric unfolded conformation of melittin at acidic pH. The ability to trap labile protons by solvent quenching makes it possible to extend amide protection studies to peptide ligands or labile protons on the surface of a protein involved in macromolecular interactions.     

A conserved glutamate residue exhibits multifunctional catalytic roles in D-fructose-1,6-bisphosphate aldolases.

The aldolase catalytic cycle consists of a number of proton transfers that interconvert covalent enzyme intermediates. Glu-187 is a conserved amino acid that is located in the mammalian fructose-1,6-bisphosphate aldolase active site. Its central location, within hydrogen bonding distance of three other conserved active site residues: Lys-146, Glu-189, and Schiff base-forming Lys-229, makes it an ideal candidate for mediating proton transfers. Point mutations, Glu-187--> Gln, Ala, which would inhibit proton transfers significantly, compromise activity. Trapping of enzymatic intermediates in Glu-187 mutants defines a proton transfer role for Glu-187 in substrate cleavage and Schiff base formation. Structural data show that loss of Glu-187 negative charge results in hydrogen bond formation between Lys-146 and Lys-229 consistent with a basic pK(a) for Lys-229 in native enzyme and supporting nucleophilic activation of Lys-229 by Glu-187 during Schiff base formation. The crystal structures also substantiate Glu-187 and Glu-189 as present in ionized form in native enzyme, compatible with their role of catalyzing proton exchange with solvent as indicated from solvent isotope effects. The proton exchange mechanism ensures Glu-187 basicity throughout the catalytic cycle requisite for mediating proton transfer and electrostatic stabilization of ketamine intermediates. Glutamate general base catalysis is a recurrent evolutionary feature of Schiff base0forming aldolases.     

Helix opening in deoxyribonucleic acid from a proton nuclear magnetic resonance study of imino and amino protons in d(CG)3.

All exchangeable protons in a short DNA helix, d(CG)3 sodium salt, have been studied by proton nuclear magnetic resonance. The cytidine and guanosine amino protons have been assigned for the first time. As a function of temperature the cytidine amino protons and the imino protons behave very similarly, their relaxation is dominated by exchange with solvent above 30 degrees C. The guanosine amino protons, however, show that helix opening can only be described by a multistate model. The most rapid process observed is probably a twist about the helix axis which lengthens or breaks the guanosine amino hydrogen bond and allows rotation of the amino group. The second fastest process is a scissor opening into the major groove which gives rise to solvent exchange with the imino and cytidine amino protons. The slowest process observed is the complete base pair opening in which the guanosine amino protons also exchange with solvent. For the ammonium salt of the oligonucleotide, a specific ammonium ion complex is observed which at low temperature may catalyze exchange of the guanosine amino protons with the protons of the ammonium ion, but retards exchange with solvent. The complex appears to be specific for the sequence d(CpG).     

Contrasting observations on buffer catalysis of guanosine amino proton exchange.

The two amino protons of 3', 5'-cyclic guanosine monophosphate are shown to differ drastically in their solvent exchange properties: One is rapidly exchanging and sensitive to buffer catalysis; the other slow and insensitive. This observation accounts for the marked contrast between stopped-flow and NMR observations on buffer catalysis of amino proton exchange in guanosine monophosphates. The amino protons of guanine compounds traverse a "fast" solvent exchange position through the process of amino rotation, which together with kinetic considerations and comparative data on adenine and cytosine compounds, supports proposals of solvent exchange mediated by events at the guanine (N-3) site, rather than the (N-7) site. Exchange does not conform to rate expressions used by different workers for amino proton exchange.     

Mechanism of hydroxylamine mutagenesis: tautomeric shifts and proton exchange between the promutagen N6-methoxyadenosine and cytidine

Whereas the amino, but not imino, tautomer of the promutagen N6-methoxyadenosine (OMe6A) forms planar associates (base pairs) with the potentially complementary uridine [Stolarski, R., Kierdaszuk, B., Hagberg, C.-E., & Shugar, D. (1984) Biochemistry 23, 2906-2913], it has now been found, with the aid of 1H NMR spectroscopic techniques, that only the imino tautomer of OMe6A base pairs with the potentially complementary cytidine. The association constant for such heteroassociates is more than an order of magnitude higher than that for autoassociates of OMe6A. The formation of heteroassociates is accompanied by a marked shift in tautomeric equilibrium of OMe6A, with an increase in the population of the amino form from 18% to as high as 44% and a corresponding decrease in the population of the imino species. Furthermore, the presence of cytidine in a solution of OMe6A appreciably enhances the rate of tautomeric exchange between the two tautomeric forms. Formation of planar heteroassociates between cytidine and the imino form of OMe6A is also accompanied by proton exchange between the cytidine NH2 and the N6-H of the amino form of OMe6A. The rate constants for this exchange and for tautomeric exchange, determined by the saturation transfer technique, have been measured at various concentrations and temperatures. A model is advanced for proton exchange that takes into account the interdependence of tautomeric exchange and proton exchange, as well as the role of auto- and heteroassociates. The relevance of these results to the molecular basis of hydroxylamine and methoxyamine mutagenesis and to the phenomenon of proton exchange in other systems is briefly discussed.     

Comparative structural analysis of cytidine, ethenocytidine and their protonated salts III. 1H, 13C and 15N NMR studies at natural isotope abundance

The 1H, 13C, 15N NMR spectra of cytidine /Cyd/, ethenocytidine /epsilon Cyd/ and their hydrochlorides /Cyd X HC1/ and /epsilon Cyd X HC1/ have been analysed to compare structural differences observed in solution with those existing in the crystalline state. The effects of ethenobridging and protonation of the hertero-aromatic base on the intramolecular stereochemistry, intermolecular interactions and electronic structure of the whole molecule are discussed on the basis of the NMR studies in DMSO solutions. Particular interest is devoted to the discussion of the conformation of the ribose ring, the presence of the intramolecular C-5'-0...H-6-C hydrogen bond, unambiguous assignment of the site of protonation, the mechanism of the 5C-H deuterium exchange in Cyd X HC1, and the intermolecular interactions in solution.     

Estimation of free energy barriers in the cytoplasmic and mitochondrial aspartate aminotransferase reactions probed by hydrogen-exchange kinetics of C alpha-labeled amino acids with solvent

The existence of the postulated quinonoid intermediate in the cytoplasmic aspartate amino-transferase catalyzed transamination of aspartate to oxaloacetate was probed by determining the extent of transfer of tritium from the C alpha position of tritiated L-aspartate to pyridoxamine 5'-phosphate in single turnover experiments in which washout from the back-reaction was obviated by product trapping. The maximum amount of transferred tritium observed was 0.7%, consistent either with a mechanism in which a fraction of the net transamination reaction proceeds through a quinonoid intermediate or with a mechanism in which this intermediate is formed off the main reaction pathway. It is shown that transfer of labeled hydrogen from the amino acid to cofactor cannot be used to differentiate a stepwise from a concerted transamination mechanism. The amount of tritium transferred is a function of the rate constant for torsional equilibration about the epsilon-amino group of Lys-258, the presumptive abstractor of the C alpha proton; the relative rate constants for hydrogen exchange with solvent versus cofactor protonation; and the tritium isotope effect on this ratio. The free energy barriers facing the covalent intermediate between aldimine and keto acid product (i.e., ketimine and possibly quinonoid) were evaluated relatively by comparing the rates of C alpha-hydrogen exchange in starting amino acid with the rates of keto acid formation. The value of theta (= kexge/kprod) was found to be 2.6 for the reaction of cytoplasmic isozyme with aspartate and ca. 0.5 for that of the mitochondrial form with glutamate.     

Reverse Action of Ribonuclease T1 Variants In ICE

Abstract

Synthesis of guanylyl(3′→5′)cytidine catalysed by RNase T1 variants (Tyr42Trp, Tyr24Trp and GluSSAla) was studied in frozen aqueous systems at-10°C and in solution at 0°C. Freezing the reaction mixture resulted in significantly enhanced dinucleoside monophosphate yields independently of the effect of mutation on substrate binding and catalytic mechanism. We assume that the protonation state of the catalytic residues is influenced by freezing, possibly due to conformational changes of the enzyme proteins.     

Hydration of Cytidine, 2′-Deoxycytidine and their Phosphate Salts in the Aqueous Solutions. A Molecular Dynamics Computer Simulation Study

Abstract

To compare the hydration pattern of the cytidine (Cyd) and 2′-deoxycytidine (dCyd) in the aqueous solutions at the level of microscopic interactions, Molecular Dynamics (MD) computer simulations have been undertaken. The results indicate that the hydration of the heterocyclic base moiety in cytidine and 2′-deoxycytidine has a hydrophobic character. None of the three potential Watson—;Crick base pair centres hydrogen bonds with the water molecules and the formation of something akin to a clathrate cage structure of water around base moieties of nucleosides in the aqueous solution is suggested. In contrast, the hydration of Cyd and dCyd sugar moieties shows a hydrophilic character and the three-dimensional networks of H-bonds involving all hydrophilic centres are formed differently around the ribose and 2′-deoxyribose. The sugar hydroxyl groups participate in the hydrogen bonding with water both as H-donor and as H-acceptor. Their donor-acceptor abilities have been evaluated and compared. The coordination numbers, the geometrical data of the first hydration shell, and the number of hydrogen bonds have been calculated. The changes in the pattern of hydration with the increased concentration of nucleosides and upon nucleoside protonation are discussed. The analysis of the pairwise interaction energies are also presented.     

The Amino 1H Resonances of Oligonucleotide Helices: d(CGCG)

Abstract

An examination of the ¹H NMR assignments and exchange properties of the amino resonances of the self-complementary tetramer, d(CGCG) was undertaken with regard to buffer effects, transfer of saturation from the water resonance and temperature dependence of amino ¹H line shape and chemical shift. The lack of buffer effect on visible exchangeable proton resonances is evidence for the stringent requirement for nucleo-base protonation at pH values below neutrality, which is greatly reduced in the helical state. For this reason, sharp resonances are observed for both Watson-Crick and non-Watson-Crick cytosine amino protons for base-paired regions. Considerations of monomeric exchange mechanisms for the cytosine and guanine amino protons formed the basis for successful assignment and isolation of their resonances in the helical state by presaturation of the water resonance at selected pH values. Preirradiation of the water resonance at pH <6 would isolate the guanine amino ¹H resonances of any self-complementary oligonucleotide, to exploit its high sensitivity as a useful proble of helix ? coil premelting.     

Solvent assistance in regiospecific disulfide formation in dimethylsulfoxide

Summary This paper describes a convenient oxidative refolding method that exploits the dual property of dimethylsulfoxide (DMSO) as an oxidant and a solvent ‘chaperon’ in assisting disulfide formation in peptides. Synthetic peptides with preferred secondary structures were used as models. For β-sheet peptides, an active fragment of fibroblast growth factor containing the tetrapeptide Arg-Ser-Arg-Arg at a reverse turn was used. A disulfide scan consisting of 16 analogs is designed in which different pairs of amino acids on the antiparallel β-sheets flanking the active determinants are replaced with cysteine. DMSO in aqueous buffer at pH 6 or 8 was found to minimize aggregation due to β-sheet formation in all 16 β-stranded peptides and provided monocyclic disulfide peptides within 7 h. In contrast, air oxidation required a significantly longer duration for completion under similar conditions, and only 8 of 16 peptides formed intramolecular disulfides. Rate accelerations at pH 8 were found in exo-disulfide formation involving peptides with amino terminal cysteine, irrespective of oxidation conditions. The exo-disulfide effect in accelerating disulfide formation may be useful for regiospecific disulfide formation. For α-helix peptides, the twodisulfide endothelin (ET) was used as a model. DMSO in combination with trifluoroethanol (TFE) was found to favor the desired bicyclic 1,4-disulfide bridged ET (1,4-ET) over the incorrectly folded 1,3-ET. Under aqueous conditions at pH 5–11, 1,4-ET to 1,3-ET was formed in the ratio of approximately 3:1, while the use of DMSO and TFE increased the ratio to 11:1. This solvent combination may stabilize an α-helical stretch found in ET and contribute to enhanced selectivity. Thus, our results show that DMSO in disulfide formation in an aqueous or helix-promoting solution may serve as an oxidant and a ‘chaperon’ solvent system to provide regiospecificity for oxidative disulfide formation.     

Kinetic analysis of protonation of a specific site on a buffered surface of a macromolecular body

The kinetics of protonation of a specific site on a macromolecular structure (micelle) in buffered solution was studied with the purpose of evaluating the effect of buffer on the observed dynamics. The experimental system consisted of the following elements: Brij 58 micelles serving as homogeneous uncharged macromolecular bodies, bromocresol green, a well-adsorbed proton detector, and 2-naphthol-3,6-disulfonate as a proton emitter in the bulk. Imidazole was the mobile buffer while neutral red, which has a high affinity for the micellar surface, served as the immobile buffer. An intensive laser pulse ejects a proton from the proton emitter, and the subsequent proton-transfer reactions are measured by fast spectrophotometric methods. The dynamics of proton pulse in buffered solution are characterized by a very rapid trapping of the discharged protons by the abundant buffer molecules. This event has a major effect on the kinetic regime of the reaction. During the first 200 ns the proton flux is rate limited by free-proton diffusion. After this period, when the free-proton concentration decayed to the equilibrium level, the relaxation of the system is carried out by the diffusion of buffer. Thus in the buffered biochemical system, at neutral pH, most of proton flux between active sites and bulk is carried out by buffer molecules--not by diffusion of free protons. Surface groups on a high molecular weight body exchange protons among them at a very fast rate. This reaction has a major role on proton transfer from a specific site to the bulk.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acid-induced exchange of the imino proton in G.C pairs.

Acid-induced catalysis of imino proton exchange in G.C pairs of DNA duplexes is surprisingly fast, being nearly as fast as for the isolated nucleoside, despite base-pair dissociation constants in the range of 10(-5) at neutral or basic pH. It is also observed in terminal G.C pairs of duplexes and in base pairs of drug-DNA complexes. We have measured imino proton exchange in deoxyguanosine and in the duplex (ATATAGATCTATAT) as a function of pH. We show that acid-induced exchange can be assigned to proton transfer from N7-protonated guanosine to cytidine in the open state of the pair. This is faster than transfer from neutral guanosine (the process of intrinsic catalysis previously characterized at neutral ph) due to the lower imino proton pK of the protonated form, 7.2 instead of 9.4. Other interpretations are excluded by a study of exchange catalysis by formiate and cytidine as exchange catalysts. The cross-over pH between the regimes of pH-independent and acid-induced exchange rates is more basic in the case of base pairs than in the mononucleoside, suggestive of an increase by one to two decades in the dissociation constant of the base pair upon N7 protonation of G. Acid-induced catalysis is much weaker in A.T base pairs, as expected in view of the low pK for protonation of thymidine.     

Effect of buffer on kinetics of proton equilibration with a protonable group

The laser-induced proton pulse generates a massive, brief, proton pulse capable of perturbing biochemical equilibria. The time resolution of the monitoring system can follow the diffusion-controlled protonation of specific sites on macromolecular bodies [Gutman, M. (1984) Methods Biochem. Anal. 30, 1-103]. In order to apply this method in enzymology, one must first evaluate how the buffer capacity of biochemical systems (substrates and proteins) will affect the observed dynamics. Unlike equilibrium measurements, where buffer is an inert component, in kinetic studies buffer modulates the observed dynamics. In this paper we analyze the effect of buffer on the dynamics of protonation in a model system. We describe the experimental technique and introduce the mathematical formalism that determines the various rate constants involved in the reaction. The analysis of the experiments indicates that in buffered solution proton flux is carried by two mechanisms: (A) proton dissociation followed by free proton diffusion; (B) collisional proton transfer between small diffusing solutes. We demonstrate how to evaluate the contribution of each pathway to the overall proton flux.