ERWIN BUNCEL, HELEN A. JOLY, AND DIANE C. YEE Department of Chemistry, Queen's University, Kingston, Ont., Canada K7L 3N6 Received December 1, PDF

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Metal ion - biomolecule interactions. Part 14.' Methylmercury and hydrogen ion catalysis of C(2)-H isotopic exchange in 1-methylhistidine ERWN BUNCEL, HELEN A. JOLY, AND DANE C. YEE Department of Chemistry,
Metal ion - biomolecule interactions. Part 14.' Methylmercury and hydrogen ion catalysis of C(2)-H isotopic exchange in 1-methylhistidine ERWN BUNCEL, HELEN A. JOLY, AND DANE C. YEE Department of Chemistry, Queen's University, Kingston, Ont., Canada K7L 3N6 Received December 1, 1988 ERWN BUNCEL, HELEN A. JOLY, and DANE C. YEE. Can. J. Chem. 67, 1426 (1989). The rate constants for detritiation from the C(2) position of 1-meth~l[2-~~]histidine have been determined in a series of aqueous buffers at 8S0C. The resulting sigmoidal rate-ph profile was indicative of a mechanism involving hydroxide ion attack on the N(3)-protonated (4) and the amino-protonated (5) forms of 1-methylhistidine, and dissection of the kinetic data allowed the extraction of the second-order rate constants for the two pathways, k and kt. The unusually large value of k' for a species not protonated at N(3) of the imidazole ring suggested the involvement of a kinetically equivalent zwitterionic form of the substrate (7). Comparison of the rate constant k with values determined previously for closely related substrates, such as histidine, 1-methylimidazole, and imidazole, led to the use of FMO theory to explain the effect of the various structural changes, e.g., the effect of methylation and a positively charged side chain on k and k'. The addition of MeHgN03 resulted in a decrease in the pseudo-first-order rate constant for dehitiation. The rate retardation was discussed in terms of two mechanisms (Schemes 2 and 3). Analysis of the data in terms of the various metal-ion-coordinated species present under the experimental conditions showed that the reactivity of the protonated substrate greatly exceeds that of the metal-coordinated species. The difference in the catalytic ability of H+ vs. MeHg+ is discussed in terms of the extent of positive charge developed on the ligating heteroatom in the ylide (carbenoid) reaction intermediate. Key words: methylmercury, 1-methylhistidine, isotopic exchange, proton transfer, metal ion catalysis. ERWN BUNCEL, HELEN A. JOLY et DANE C. YEE. Can. J. Chem. 67, 1426 (1989). On a dktermink les constantes de vitesse de la dktritiation h partir de la position C(2) de la rn~th~l-l[~~-2]-histidine dans une skrie de solutions tampons?i 85 C. Le profil sigmoidal de la courbe de vitesse en fonction du ph obtenue, tkmoigne 'existence d'un mkcanisme faisant intervenir l'attaque de l'ion hydroxyle sur les formes protonkes en N(3) (4) et amino-protonke (5) de la mkthyl-1-histidine. Le dkpouillement des donnkes cinktiques permet d'extraire les constantes de vitesse d'ordre deux des deux sites d'attaque, k et k'. La grande valeur inhabituelle de k' pour les espkces non protonkes au niveau N(3) du cycle imidazole suggkrent 'intervention d'une forme zwitterionique cinktiquement kquivalente du substrat (7). La comparaison de la constante de vitesse k avec les valeurs dkterminkes antkrieurement pour des substrats trks apparentks cornme l'histidine, le mkthyl-1-imidazole et l'imidazole, conduit?i utiliser la thkorie des OML pour expliquer l'effet des divers changements structuraux, par exemple, l'effet d'une mkthylation et d'une chaine latkrale chargke positivement sur les valeurs de k et de k'. L'addition du MeHgN03 conduit h une diminution de la constante de vitesse de pseudo ordre un de la dktritiation. On discute de la diminution de la vitesse en fonction de deux mkcanismes (schkmas 2 et 3). L'analyse des donnkes en fonction des diverses espi?ces ion mktallique-coordonnk prksentes dans les conditions exp6rimentales dkcrites, montre que la rkactivitk du substrat proton6 exckde de beaucoup celle des espkces mktalliques coordonnkes. On discute de la diffkrence de pouvoir catalytique de H+ versus MeHg+ en fonction du taux de charge positive dkveloppke au niveau de l'hktkroatome jouant le r6le de ligand dans l'intermkdiaire (carbknoide) de rkaction de l'ylide. Mots clks : mercure-mkthyle, mtthyl-1-histidine, tchange isotopique, transfert de proton, catalyse d'ion mttallique. [Traduit par la revue] Histidine (1) is one of the most important amino acids found in biological systems and is present at the active site of enzyme systems such as ribonuclease and a series of serine proteases including a-chymotrypsin, trypsin, and subtilisin (1). n addition, histidine is an essential constituent of several metalloproteins including haemoglobin, myoglobin, and histidine deaminase, presumably because of the availability of the electron pair on N(3) of the heteroaromatic ring to bind transition-metal cations (2). n imidazole (2), the heteroaromatic component of histidine, the C(2)-H flanked by the two heteroatoms, is relatively labile and upon prolonged heating in D20 or tritiated water will undergo isotopic exchange (3). This property has proven useful in probing biological systems and was used to identify the metal-coordinated histidine residues of the enzymes p-lactamase 1 and superoxide dismutase (4, 5). The optimal use of labelled biochemical reactants for elucidating accurate mechanistic information about an enzymatic process requires that one clearly understands the factors that affect tritium and deuterium loss, such as variation in ph, 'Part 13: ref. 26. coo- + temperature, and the presence of various metal ions. n one of the earliest reported studies concerning the effect of metal ions on isotopic hydrogen exchange in heterocyclic compounds, with 1-methyltetrazole as the substrate, Olofson et al. (6) found that complexation with the transition metal ions Cu2+ and Zn2+ enhanced the rate of C(5)-H exchange considerably. Based on a mechanism involving hydroxide ion attack on a 1:l Mn+-lmethyltetrazole complex, it was found that metal complexation was 6 to 403 times less effective for exchange than the path involving the conjugate acid, but 5 to 294 times faster than the path involving the neutral substrate. Jones and co-workers (7) studied the effect of metal ions on the rate of detritiation of [2-3H]imidazole at 85 C and ph 5.70 and found without exception that addition of metal ions caused rate retardation. The effectiveness of the metal ion increases in the order Cu2+ zn2+ ;= Ni2+ MeHg+. The results suggested that formation of metal complexes reduced the concentration of the active kinetic species, namely the conjugate acid of imidazole. Martin and co-workers (8) extended the study of the effect of metal ions on reactivity of the isotopic hydrogen exchange reaction to 1-methylhistidine (la). The rate constant for deuteration of the 2: 1 complex of 1-methylhistidine and pd2+ was measured in D20 at 61 C at various ph values ranging from 11.3 to 12.0 using NMR spectroscopy. The results showed that hydroxide ion attack occurs and times faster for the amino-protonated form (la) and the 1-methylhistidine species protonated both on N(3) of the imidazole group and the amino group of the side chain, than for the pd2+ complex. Comparison of the rate constants in this fashion gives a quantitative measure of the effect of metal ions on reactivity. A significant decrease in the detritiation rate constants observed on addition of copper ions to an aqueous solution of 1-methyl[8-~~]guanosine (9) implicates N(7) of l-methyl- [8-3~]guanosine as the site of metal complex formation, since N(l) and N(9) positions are blocked by a methyl group and a ribose group, respectively. Metal ions added to the reaction mixture compete with H+ for N(7) of the methylated purine derivatives and, as a result, the rate of isotopic exchange in the presence of metal ion depends upon the rate of hydroxide ion attack on both the protonated substrate (BH+) and the metalcomplexed (BM +) species. The ratio of the second-order rate constant for hydroxide ion attack for BH+ to that for BMn+, k and kc, respectively, was found to be 1025 indicating that Cu2+ was not as effective as the proton in accelerating the rate of detritiation. However, a comparison of kc, to k,, the second-order rate constant for OH- attack on neutral 1-methyl[8-3~]guanosine (9), shows that Cu2+ coordinated to N(7) does in fact catalyze the reaction (kcu/kn = 10~.~). The ratio k,/k, has been called the metal-activating factor (10). n a study of the rates of detritiation of l-methyl[8-3h]inosine in the presence of cu2+, zn2+, and Ag+ ions, the metal ions were found to influence the rate of isotopic exchange in very different ways (10). For example, Zn2+ had virtually no effect on the rate of detritiation while Ag+ was found to suppress the rate of exchange to an even greater degree than Cu2+. This observation is consistent with the ability of the metal ion to coordinate with the N(7) centre. Ag+, as a soft metal ion, binds more strongly to nitrogen centres than do the two borderline metal ions, zn2+ and Cu2+. Since Ag+ binds most strongly, the complex will be in higher concentration than the complexes of Zn2+ and cu2+, and thus Ag+ causes greatest rate retardation. A similar treatment (9) to that used for 1-methylguanosine results in a metal-activating factor (k,/kn) of lo7 for Cu2+ and lo5 for Ag+, and a proton activation factor (klk,) of lo3 and lo4.' for cu2+ and Ag+, respectively. From all the metal ion studies carried out so far, it can be concluded that metal ions significantly retard the rate of C(2)-H exchange with respect to the N(3)-protonated species (7, 8). Thus, metal ions are not as effective catalysts as is the proton in promoting C(2)-H exchange. n the present study, the 1-methylhistidine - methylmercury nitrate system has been chosen on the basis of several considerations. First, methylmercury(1) is known to form relatively strong complexes with N-containing ligands, compared with transition-metal salts of Zn2+, Cu2+, etc. (2, 11). Secondly, BUNCEL ET AL the preferred coordination number of MeHg' is one, unlike transition-metal ions such as cu2+, which coordinate 1-6 ligands depending upon the experimental conditions. Thirdly, the MeHg+ hydroxides that form are soluble at relatively high ph, allowing rate constants to be measured at higher ph values (11). t is worth noting, however, that hydroxide ion is a very effective competitor for MeHg+ and a delicate balance exists between the ph value that is most appropriate to monitor a change in the magnitude of the rate constant and the ph value that minimizes the competition of hydroxide ion and l-methylhistidine for MeHg+. The similarity in structure between 1-methylhistidine (la) and histidine (1) makes it an attractive substrate to study. Blocking N(l) with a methyl group reduces the potential MeHg+ binding sites from 4 to 3, thereby significantly reducing the complexity of the kinetic analysis. The first section of the paper describes the kinetic analysis of the data collected for 1-methyl[2-3~]histidine (la) in aqueous buffers at 85 C so as to obtain a rate-ph profile. With that necessary information, results were obtained for the detritiation of l-methy:1[2-3h]histidine in a series of MeHgN03 solutions varying in concentration from 0 to M. The observed decrease in pseudo-first-order rate constant with increase in metal-ion concentration can be accounted for by mechanisms involving rate-determining triton abstraction from C(2) of N(3)-protonated or metal-coordinated species to give an ylide (carbenoid) intermediate, which is then rapidly protonated (12). The detailed discussion involves consideration of ionized states in the side chain (R') and the relative effectiveness of H+ vs. M+ in exchange (see Schemes 2 and 3). Experimental Preparation of 1 -methyl-l-histidine 1-Methyl-L-histidine dihydrochloride was prepared by the three-step sequence outlined by Noordarn et al. (13a). The 'H NMR spectrum in D20 compared well to that previously reported (6 ppm: 3.38 (2H, 8-peak multiplet, CH,), 3.89 (3H, singlet, NMe), 4.24 (H, triplet, NHzCH), 7.41 (H, singlet, CCHN), 8.66 (lh, singlet, NCHN). The R spectrum showed an absorption at 1709 cm-' characteristic of a carboxylic acid. The presence of the ammonium salt is evidenced by several absorption bands: a medium broad band at 3400 cm-, a group of strong bands between 1575 cm-' and 1600 cm-', and one strong absorption found at 1495 cm-. Finally, ascending paper chromatography was performed on the dihydrochloride product against L-histidine as a standard. The two compounds were spotted (5 pg) on cellulose paper and developed in a chromatography tank saturated with 0.1 M a-picoline for 110 rnin. The dried paper was sprayed with 1428 CAN. J. CHEM. VOL. 67, 1989 ninhydrin and heated in an oven for 15 min at which time purple spots pertaining to each of the two compounds appeared. The 1-methyl-Lhistidine dihydrochloride (Rf = 0.868) was found to migrate further than the L-histidine (Rf = 0.814) consistent with that previously reported (13 b). 1-Methyl-L-histidine dihydrochloride was desalted using cation exchange chromatography. The 1-methyl-L-histidine dihydrochloride (0.425 g) dissolved in 1.5 ml of 2 N HCl was loaded onto a Dowex 50 X ( mesh) column (1 cm diameter x 13 cm high). Deionized distilled water was eluted through the column to remove all chloride ions. This was continued until addition of AgN03 showed no evidence of AgCl precipitation. To remove the 1-methyl-L-histidine as the free base from the column, 4 N NH40H was used as an eluant. Water and ammonia were removed by rotoevaporation. The residue was dried under vacuum overnight (0.208 g, 69.8% yield, mp C (lit. (12) mp C)). deionized, distilled water in a 100-mL volumetric flask. Potassium nitrate (dried at 100OC) was added to maintain constant ionic strength. Adjustment of the ph of the metal-ion solutions The metal-ion solution was placed in a reaction vessel designed to carry out potentiometric titrations, which was fitted with a cover with several openings. A combination ph electrode was inserted into one of the openings while the Teflon tubing attached to a microburette was inserted through a small hole punched through a rubber stopper; all other openings were sealed. A stining bar was used throughout the ph-adjustment procedure to ensure efficient mixing. Very small quantities of 5% HN03 or 0.01 M NaOH were delivered to the metal salt solution to a desired ph with the aid of the microburette of a Metrohm-Herisau automatic titrator. After addition of the final aliquot of acid or base, the solution was allowed to stir for ca. 3-5 min before the ph reading was recorded. Synthesis of 1 -methyl[2-3~]-~-histidine Kinetic measurements l-ritiated water (30,,L, 150 m ~i) was added via a syringe to freshly The rate of detritiation of 1-methyl[2-3H]histidine was measured by purified l-methyl-l-histidine (97.75 mg) in a heavy-walled glass monitoring the increase in radioactivity of the reaction mixture with ampode. The glass ampoule was capped with a rubber septum, its time (la). The kinetic Stock solution (25 FL) was dispensed by means contents frozen in liquid nitrogen, and the evacuated. After of an automatic pipette to 20 ml of buffer or metal-ion solution sealing, the ampoule was immersed in an oil bath at 85~C for 17 days, contained in a 50-mL round-bottom flask and equilibrated for no less at the end of period the ampoule was opened, ml of distilled than 30 min at 85 C. The mixture was shaken for s. Aliquots of water was added to exchange labile tritium, and the solvent was 1 ml were removed at various times (to... tn) with either a ~ohr removed by lyophilization. Addition of 1-m~ portions of water was pipette or an Ependorf automatic pipette and delivered to a 50-mL continued until the activity of the lyophilized water was found to be round-bottom flask containing 0.1 g of sodium chloride. (TO prevent insignificant. fie total activity of the substrate was approximately sublimation of 1-methylhistidine, the B14 joint of the freeze drying 14 mci. The material was yellowish brown in colour tic analysis bends was lightly plugged with glass wool.) ~t this ~ointhe time indicated two or three other impurities, which were removed as on the stopclock was read and recorded. The round-bottom flask was follows. immediately dipped into a Dewar flask containing liquid nitrogen for using a preparative silica gel plate, approximately mg (7 mci) quenching the reaction and rotated so as to cause the solution to freeze of crude material in distilled water was spotted along the baseline of the a thin On the inner of the flask. Lyophilization of the plate. The plate was developed in a closed tank containing solvent quenched samples enabled separation of the water from the substrate. (6:6:4:1 CH30H:pyridine:H20:glacial acetic acid) and equilibrated A the water from the was withdrawn overnight at room temperature. Development of the plate required 3 h with a 0.1-mL Mohr pipette and transferred to a 7-mL plastic liquid 35 min. scintillation vial containing 6 ml of Unisolve E or Beckman HP liquid under uv light, the band pertaining to the l-methyl[2-3~l-lscintillator. The kinetic sample was assayed for tritium (C,) on histidine was marked. h hi^ band of silica gel was scraped off the plate a Beckman LS5801 liquid scintillation counter. Subsequent kinetic and placed in a small Erlenmeyer flask. Distilled water (3 X 3 ml) was were treated in the same fashion' The infinity reading (C,), i.e., the radioactivity transferred to the added to extract the substrate. After filtration, water was removed by lyophilization. The total activity was found to be = 1.2 mci. aqueous solution when the exchange reaction had reached completion, A kinetic stock solution was prepared by transferring the tritiated was determined by measuring the disintegrations per minute of 0.1 ml of the reaction mixture in 6 ml of Unisolve E or Beckman HP liquid material to a test tube containing 1 ml of distilled water. The tube was scintillator. stoppered, sealed with Parafilm, and stored in the refrigerator until needed. Analysis of kinetic data Preparation of buffer solutions First-order kinetics Potassium hydrogen phthalate and sodium borate buffers were used The first-order rate constants, kobs, for the detritiation of 1- for kinetic measurements carried out at low and intermediate PH methyl[2-3h]histidine in aqueous buffers and metal-salt solutions were ranges, respectively, while sodium hydroxide solutions were used for extracted from the slopes ( kobs) of the plots of log (C- - Cr) measurements at higher ph values. vs. time. nitial rates method Preparation of methylmercury nitrate solutions (14) For slow reactions, i.e., when tl12 was greater than 6 h, an initial Methylmercury chloride (3.75 g) was suspended in 25 ml of rates method was used to determine the first-order rate constant for distilled, deionized water in an Erlenmeyer flask. To this was added a the detritiation of 1-methyl[2-3H]histidine. This method required that solution containing 2.54 g of AgN03 in 25 ml of water. The flask was C, and the corresponding time be collected for no more than 5% of stoppered, covered in aluminum foil, and allowed to stir in the dark for reaction. A plot of C, vs. time results in a straight line with a slope 6 days. At this point the solution was filtered through a sintered glass equal to kobs/cm. funnel and the AgCl resulting from the ligand exchange was collected on the filter. To ensure the absence of free Ag+ ions, 0.4 g of Results and discussion methylmercury chloride was added to the filtrate, which was refiltered c(~)-h exchange in the absence of metal ion after stining for ca. 2 h. The filtrate was transferred to a beaker The rate constants for the detritiation of l-methyl[2-3~~and allowed to evaporate in the fume cupboard until a solid residue histidine (la) in aqueous buffer solutions at 850C are listed remained. The residue was recrystallized from hot CCL. Upon cooling, white crystals formed and were collected and dried in vacuo in and shown graphically in Figure in the Of overnight, yield ca. 2.0g. a rate-ph profile for the ph range Also included A series of solutions ranging from 0 to M we
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