1H-NMR study on the tautomerism of the imidazole ring of histidine residues: II. Microenvironments of histidine-12 and histidine-119 of bovine pancreatic ribonuclease a

The NMR titration curves of proton chemical shifts were observed for the C₂ protons of histidine residues in intact bovine pancreatic RNAase A (EC 3.1.27.5) and carboxyalkylated RNAase A. By comparing the methyl region of NMR spectra, the 250–340 nm region of circular dichfoic spectra, and the NMR titration curves of tyrosine ring protons among intact and modified RNAase A, it was ascertained that the carboxyalkylation of histidine residues at position 12 or 119 did not make any appreciable conformational changes to RNAase A. With the pK values determined for intact and modified RNAase A, the microscopic pK values and molar ratios of tautomers were estimated for His-12 and His-119 by means of the procedure described in the preceding paper. The estimated microscopic pK values of tautomers were 6.2 for the N₁-H tautomer of His-12, more than 8 for the N₃-H tautomer of His-12, 7.0 for the N₁-H tautomer of His-119, and 6.4 for the N₃-Hf tautomer of His-119, respectively. These values were interpreted in terms of the microscopic environments surrounding the histidine residues. The microscopic structure estimated in the present study was discussed, comparing it with those from X-ray crystallography and hydrogen-tritium (or hydrogen-deuterium) exchange technique.     

1H-NMR study on the tautomerism of the imidazole ring of histidine residues. I. Microscopic pK values and molar ratios of tautomers in histidine-containing peptides

The 1H-NMR titration curves of chemical shifts versus pH were observed for imidazole, N1-methylimidazole, L-histidine, N1-methyl-L-histidine, N3-methyl-L-histidine, and other related compounds. With these results, the macroscopic pK values of these compounds were determined by a computer curve-fitting for a simple dissociation sequence. From the pK values of imidazole and N1-methylimidazole, the perturbation for the pK of the imidazole ring due to the substitution of a proton with a methyl group was estimated as -0.21 pH unit. The microscopic pK values of the individual tautomers of the imidazole ring were estimated with the pK values of N1-methyl-L-histidine, N3-methyl-L-histidine, and perturbation due to methyl substitution. The estimated pK values were 6.73 for the N1-H tautomer and 6.12 for the N3-H tautomer. These values were in good agreement with those obtained using carboxymethyl derivatives instead of methyl derivatives. Furthermore, the macroscopic pK value (6.02) calculated using the estimated microscopic pK values agreed with that (6.03) observed for the imidazole ring of L-histidine. Thus the method in this work was indicated to be self-consistent. The microscopic pK values of tautomers were also obtained for N alpha-acetyl-L-histidine and N alpha-acetyl-L-histidine methylamide. The molar ratios of tautomers were calculated on the basis of the microscopic pK values of tautomers. The intrinsic (or unperturbed) pK value of imidazole ring and perturbations due to the CO2- and NH3+ were obtained for each of the N1-H and N3-H tautomers.     

Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. III. Mutual electrostatic interaction between histidine residues 12 and 119.

The two adjacent active site histidine residues of bovine pancreatic ribonuclease A (histidine-12 and -119) yield proton magnetic resonance titration curves having Hill coefficients significantly less than unity (0.7 and 0.8, respectively). Three models postulating interactions with other titrating groups in the molecule have been used to approximate these anomalous experimental titration curves. Very good agreement with the data was obtained with models postulating mutual electrostatic interaction between histidine-12 and -119. The additional low pH perturbation of the chemical shift of the C(2)-H peak (but not the C(4)-H peak) of histidine-12 is attributed to a local conformational change with a pHmid of about 3.5.     

1H-NMR studies on the binding subsites of bovine pancreatic ribonuclease A

The titration curves of the C-2 histidine protons of an RNAase derivative (a covalent derivative obtained by reaction of bovine pancreatic RNAase A (EC 3.1.27.5) with 6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate) were studied by means of 1H-NMR spectroscopy at 270 MHz. The interaction of natural (5'AMP, 5'GMP, 5'IMP) and halogenated purine mononucleotides (cl6RMP, br8AMP) with RNAase A was also monitored by using the same technique. The slight change observed in the pK values of the active centre histidine residues of the RNAase derivative, with respect to those in the native enzyme, can be considered as evidence that the phosphate of the label does not interact directly either with His-12 or 119 in the p1 site, but the p2 site as proposed previously (Parés, X., Llorens, R., Arús, C. and Cuchillo, C.M. (1980) Eur. J. Biochem. 105, 571--579). Lys-7 and/or Arg-10 are proposed as part of the p2 phosphate-binding subsite. The pK values of His-12 and 119 and the shift of an aromatic resonance of the native enzyme found on interaction with some purine nucleotides, can be interpreted by postulating that the interaction of 5'AMP, 5'GMP and 5'IMP takes place not only in the so-called purine-binding site B2R2p1 but also in the primary pyrimidine-binding site B1R1 and p0 of RNAase A.     

Sequential 1H-NMR assignment and solution structure of bovine pancreatic ribonuclease A

Assignments for 1H-NMR resonances of most of the residues of bovine pancreatic ribonuclease (RNase A) have been obtained by sequence-specific methods. Identification and classification of spin systems have been carried out by two-dimensional phase-sensitive correlated spectroscopy (360 MHz) and single relayed coherence transfer spectroscopy. Sequence-specific assignments have been achieved by phase-sensitive two-dimensional nuclear Overhauser effect spectroscopy. To overcome the problem of spectral overlap use has been made of (a) an exhaustive analysis of partly exchanged RNase A (spectra in D2O), (b) a comparison with the subtilisin-modified enzyme (RNase S) and (c) small spectral perturbations caused by changes in pH and temperature. The secondary structure elements have been identified from the observed sequential, medium and long-range nuclear Overhauser effects together with data from amide-exchange rates. All information collected leads to the conclusion that the crystal and the solution structures are closely similar.     

Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. II. pH and inhibitor-induced conformational transitions affecting histidine-48 and one tyrosine residue of ribonuclease A.

The microenvironment of histidine-48 of bovine pancreatic ribonuclease A was investigated by proton magnetic resonance spectroscopy (1H NMR) using partially deuterated enzyme in which resolution of the C(2)-H resonance of histidine-48 was simplified. The NMR titration curves at 100 and 250 MHz of histidine-48 of ribonuclease A are discontinuous both for the enzyme alone in 0.3 M chloride and for its complex with cytidine 3'-phosphate. This suggests that titration of histidine-48 occurs only as the result of a slow conformational transition. The sum of the peaks corresponding to histidine-48 in the acid-stable and base-stable forms of the enzyme is less than one proton in the transition region, which indicates that there exists at least one intermediate conformational form of the enzyme. The transition from the acid-stable form to an intermediate form has a pHmid of 5.6, and the transition from an intermediate form to the base-stable form has a pHmid of 6.9. In ribonuclease S and in ribonuclease A in the presence of 0.3 M acetate, the titration curve of histidine-48 is continuous, and the area of the peak is uniform throughout the titration. Proton NMR difference spectra at 100 and 250 MHz reveal a pH-induced conformational change with a pHmid of 5.7 that affects the chemical shift of a single tyrosine residue. This conformational transition is absent in ribonuclease S and is altered in ribonuclease A by the presence of either acetate or cytidine 3'-monophosphate. It is postulated that the same conformational transition is responsible for both the tyrosine perturbation and the disappearance of the histidine-48 peak observed in the acid-stable form of the enzyme. It is proposed that the perturbed tyrosine is tyrosine-25. The transition with pHmid 5.6 is attributed to dissociation of aspartic acid-14, and the transition with pHmid 6.9 is assigned to dissociation of histidine-48. A peak in the aromatic region that moves upfield on addition of the competitive inhibitor cytidine 3'-monophosphate is assigned to a tyrosine, and evidence is presented that this tyrosine is tyrosine-25. Inhibitor binding appears to induce a conformational change in the histidine-48/tyrosine-25 region which is remote from the active site.     

Effect of bovine seminal ribonuclease and bovine pancreatic ribonuclease A on bovine oocyte maturation

Bovine seminal ribonuclease (BS-RNase) contains the MxM (noncovalent dimer) and M=M (free monomer) in constant ratio. The aim of this work was to evaluate the effect of BS-RNase, its monomer and dimer forms, and also various mutants of this enzyme on meiotic completion in cattle oocytes. It was found that BS-RNase has irreversible effects on the meiotic maturation of bovine oocytes in vitro, particularly on the completion of meiosis. The effect of BS-RNase is dose-dependent. In medium supplemented with 1 microg/ml, the results were comparable with those of the control (70% MII oocytes after 24 hr of culture). Whereas 5 microg/ml reduced the number of MII oocytes to 50%, 10 and 25 microg/ml arrested this process completely. The MxM form and RNase A at 5 microg/ml inhibited the maturation rate by 71 and 48%, respectively, but a less significant effect was observed for the M=M form, or the carboxymethylated monomers MCM31 and MCM32 (21%, 16%, and 42% MII oocytes, respectively, in comparison with control). These data demonstrate that bovine ribonucleases can have variable detrimental effects on the maturation of bovine oocyte. J. Exp. Zool. 287:394-399, 2000.     

Nuclear magnetic resonance titration curves of histidine ring protons. Conformational transition affecting three of the histidine residues of ribonuclease.

NMR titration curves are reported for the 4 histidine residues of ribonuclease A in sodium acetate and for ribonuclease S in sodium acetate, phosphate, and sulfate solutions. Evidence is presented that the imidazole side chain of histidine residue 48 undergoes a conformational change, probably also involving the carboxyl side chain of aspartic acid residue 14. This group is considered to be responsible for the low pH inflection with pKa 4.2 present in the NMR titration curve of the C-2 proton resonance of histidine 48. The NMR titration curves of the active site histidine residues 12 and 119 also exhibit inflections at low pH values, although there is no carboxyl group within 9 A of the imidazole side chain of histidine residue 12 in the structure of ribonuclease S determined by x-ray crystallography (Wyckoff, H. W., Tsernoglou, D., Hanson, A. W. Knox, J. R., Lee, B., and Richards, F. M. (1970) J. Biol. Chem. 245, 305-328). Curve fitting was carried out on 11 sets of NMR titration data using a model in which the 3 histidine residues 12, 119, and 48 are assumed to be affected by a common carboxyl group. The results obtained indicate that such a model with fewer parameters gives as good a representation of the data as the model in which each histidine residue is assumed to interact separately with a different carboxyl group. Therefore, it is concluded that the ionization of aspartic acid residue 14 is indirectly experienced by the active site histidine residues through the conformational change at histidine 48. A model assuming mutual interaction of the active site histidine residues does not account for the low pH inflections in these curves.     

The aromatic residues of bovine pancreatic ribonuclease studied by 1H nuclear magnetic resonance.

1. The aromatic proton resonances in the 360-MHz 1H nuclear magnetic resonance (NMR) spectrum of bovine pancreatic ribonuclease were divided into histidine, tyrosine and phenylalanine resonances by means of pH titrations and double resonance experiments. 2. Photochemically induced dynamic nuclear polarization spectra showed that one histidine (His-119) and two tyrosines are accessibly to photo-excited flavin. This permitted the identification of the C-4 proton resonance of His-119. 3. The resonances of the ring protons of Tyr-25, Tyr-76 and Tyr-115 and the C-4 proton of His-12 were identified by comparison with subtilisin-modified and nitrated ribonucleases. Other resonances were assigned tentatively to Tyr-73, Tyr-92 and Phe-46. 4. On addition of active-site inhibitors, all phenylalanine resonances broadened or disappeared. The resonance that was most affected was assigned tentatively to Phe-120. 5. Four of the six tyrosines of bovine RNase, identified as Tyr-76, Tyr-115 and, tentatively, Tyr-73 and Tyr-92, are titratable above pH 9. The rings of Tyr-73 and Tyr-115 are rapidly rotating or flipping by 180 degrees about their C beta--C gamma bond and are accessible to flavin in photochemically induced dynamic nuclear polarization experiments. Tyr-25 is involved in a pH-dependent conformational transition, together with Asp-14 and His-48. A scheme for this transition is proposed. 6. Binding of active-site inhibitors to bovine RNase only influences the active site and its immediate surroundings. These conformational changes are probably not connected with the pH-dependent transition in the region of Asp-14, Tyr-25 and His-48. 7. In NMR spectra of RNase A at elevated temperatures, no local unfolding below the temperature of the thermal denaturation was observed. NMR spectra of thermally unfolded RNase A indicated that the deviations from a random coil are small and might be caused by interactions between neighbouring residues.     

Identification of histidine-119 as the target in the site-specific inactivation of ribonuclease A by ferrate ion.

Ferrate ion, a powerful oxidant which is an analog of orthophosphate ion, has previously shown some promise as a site-specific probe of enzymes which interact with phosphate compounds. In order to explore the general applicability of this reagent, it has been tested against ribonuclease A, an enzyme whose structure and active center have been well described. Treatment with a molar ratio of ferrate to enzyme of less than 20 leads to a loss of 87% of the activity. The known competitive inhibitors, 2'-cytidylic acid, inorganic pyrophosphate, and orthophosphate all protect the enzyme from inactivation. Inactivation is accompanied by a loss of the capacity to bind 2'-cytidylic acid. Ferrate inactivation at pH 5.0 is accompanied by the modification of only one amino acid. The amino acid which was identified by amino acid and sequence analyses of peptide fragments obtained by cyanogen bromide treatment and selective proteolysis proved to be histidine-119, whose essential role at the active center has long been established.     

Nuclear magnetic resonance titration curves of histidine ring protons. A direct assignment of the resonances of the active site histidine residues of ribonuclease.

One of the four titrating histidine ring C-2 proton resonances of bovine pancreatic ribonuclease has been assigned to histidine residue 12. This was accomplished by a direct comparison of the rate of tritium incorporation into position C-2 of histidine 12 of S-peptide (residues 1 to 20) derived from ribonuclease S, with the rates of deuterium exchange of the four histidine C-2 proton resonances of ribonuclease S under the same experimental conditions. The same assignment was obtained by a comparison of the NMR titration curves of ribonuclease S, the noncovalent complex of S-peptide and S-protein (residues 21 to 124) with the results for the recombined complex in which position C-2 of histidine 12 was fully deuterated. The second active site histidine resonance was assigned to histidine residue 119 by consideration of the NMR titration results fro carboxymethylated histidines and 1-carboxymethylhistidine 119 ribonuclease. This assignment is a reversal of that originally reported, and has important implications for the interpretation of NMR titration data of ribonuclease.     

Proton-magnetic-resonance spectroscopic study of the histidine residues of bovine alpha-lactalbumin.

A study of the three histidine residues of bovine alpha-lactalbumin has been made using proton magnetic resonance (PMR) spectroscopy in order to obtain information on their environments in the protein and thereby to test in part the previously proposed structure. PMR titration curves are obtained for the H-4 resonances using difference spectroscopy and for the H-2 resonances and the 1-H-2-H exchange rates of the H-2 protons have been measured. The assignment of resonances to particular histidine residues is achieved by utilising their selective reaction with iodoacetate in conjunction with a PMR study of the carboxymethylation of alpha-N-acetyl-L-histidine. The H-2 and H-4 resonances labelled 1, 2 and 3 starting from the downfield end of the spectrum are assigned to histidine residues 107, 68 and 32 respectively. Their apparent pK values at low ionic strength and 20 degrees C are 5.78, 6.49 and 6.51 respectively. The experimental results on two histidine residues are consistent with the predictions of the proposed structure, which indicate that histidine-68 is an external residue and histidine-32 is partially buried and in the vicinity of aromatic residues. The experimental data on histidine 107 can also be rationalised with less certainty in terms of the proposed structure, which indicates a partially buried residue that may be involved in hydrogen bonding.     

Assignment of the imidazole ring nitrogen protons of histidine 48 in the proton NMR spectrum of ribonuclease A in water solution.

Several exchangeable resonances, designated a, b, c and d are observed in the 11-14 ppm (from 2,2-dimethyl-2-silapentane-5-sulfonate) region of the proton spectrum of ribonuclease A in water solution. We describe a number of lines of evidence suggesting the assignment of peaks b and c to the N1 and N3 protons of His 48, which occupies an interior position in the protein remote from the active site. This evidence includes the observation that the binding of Cu(II) and 3'-CMP (cytidine 3'-monophosphate) has no effect on these resonances. Further evidence includes pH titration data showing a pKa of approx. 2 for these protons, solvent exchange rates in the native state and with disulfide bridges IV-V and III-VIII cleaved, the observation of the carboxymethylated enzymes CM-His12-RNAase A and CM-His119-RNAase A, and of the modified enzymes Des(1-21)-RNAase A (S-protein) and Des(119-124)-RNAase A.     

1H nuclear magnetic resonance titration curves and microenvironments of aromatic residues in bovine pancreatic ribonuclease A

The aromatic region of the NMR spectrum of bovine pancreatic ribonuclease A was analyzed in order to clarify the nature of the microenvironments surrounding the individual histidine, tyrosine, and phenylalanine residues and the interactions with inhibitors. The NMR titration curves of ring protons of six tyrosine and three phenylalanine residues as well as four histidine residues were determined at 37 degrees C between pH 1.5 and pH 11.5 under various conditions. The titration curves were analyzed on the basis of a scheme of a simple proton dissociation sequence and the most probable values were obtained for the macroscopic pK values and intrinsic chemical shifts. The microenvironments surrounding the residues and the effects of inhibitors are discussed on the basis of these results. Based on the titration curves of ring protons, the six tyrosine residues were classified into the following four groups: (1) titratable and different chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (2) titratable but similar chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (3) not titratable and different chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residues), and (4) not titratable and similar chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residue). The resonance signals of ring protons were tentatively assigned to tyrosine and phenylalanine residues. The NMR titration curves of His-48 ring protons were continuous in solution containing 0.2 M sodium acetate but were discontinuous in solution containing 0.3 M NaCl because the NMR signals disappeared at pH values between 5 and 6.5. The effects of addition of formate, acetate, propionate, and ethanol were investigated in order to elucidate the mechanism of the continuity of the titration curves of His-48 in the presence of acetate ion. The NMR signal of His-48 C(2) protons was observed at pH 6 in the presence of acetate and propionate ions but was not observed in the presence of formate ion or ethanol. This indicated that both the alkyl chain and the anionic carboxylate group are necessary for the continuity of the titration curves of His-48 ring protons. Based on the results, the mechanism of the effects of acetate ion is discussed.     

Two histidine residues are essential for ribonuclease T1 activity as is the case for ribonuclease A

Ribonuclease T1 (RNase T1, EC 3.1.27.3) is a guanosine-specific ribonuclease that cleaves the 3',5'-phosphodiester linkage of single-stranded RNA. It is assumed that the reaction is generated by concerted acid-base catalysis between residues Glu-58 and His-92 or His-40. From the results of chemical modification and NMR studies, it appeared that the residue Glu-58 was indispensable for nucleolytic activity. However, we have recently demonstrated that Glu-58 is an important but not an essential residue for catalytic activity, using the methods of genetic engineering to change Glu-58 to Gln-58 etc [Nishikawa, S., Morioka, H., Fuchimura, K., Tanaka, T., Uesugi, S., Ohtsuka, E., & Ikehara, M. (1986) Biochem. Biophys. Res. Commun. 138, 789-794]. In the present paper, we report that mutants of RNase T1 with residue Ala-40 or Ala-92 have almost no activity, while mutants that contain Ala-58 retain considerable activity. These results show that the two histidine residues, His-40 and His-92, but not Glu-58, are indispensable for the catalytic activity of the enzyme. We propose a revised reaction mechanism in which two histidine residues play a major role, as they do in the case of RNase A.     

Nuclear-magnetic-resonance study of the histidine residues of S-peptide and S-protein and kinetics of 1H-2H exchange of ribonuclease A.

1H NMR spectroscopy at 100 MHz was used to determine the first-order rate constants for the 1H-2H exchange of the H-2 histidine resonances of RNase-A in 2H2O at 35 degrees C and pH meter readings of 7, 9, 10 and 10.5. Prolonged exposure in 2H2O at 35 degrees C and pH meter reading 11 caused irreversible denaturation of RN-ase-A. The rate constants at pH 7 and 9 agreed reasonably well with those obtained in 1H-3H exchange experiments by Ohe, J., Matsuo, H., Sakiyama, F. and Narita, K. [J. Biochem, (Tokyo) 75, 1197-1200 (1974)]. The rate data obtained by various authors is summarised and the reasons for the poor agreement between the data is discussed. The first-order rate constant for the exchange of His-48 increases rapidly from near zero at pH 9 (due to its inaccessibility to solvent) with increase of pH to 10.5 The corresponding values for His-119 show a decrease and those for His-12 a small increase over the same pH range. These changes are attributed to a conformational change in the hinge region of RNase-A (probably due to the titration of Tyr-25) which allows His-48 to become accessible to solvent. 1H NMR spectra of S-protein and S-peptide, and of material partially deuterated at the C-2 positions of the histidine residues confirm the reassignment of the histidine resonances of RNase-A [Bradbury, J. H. & Teh, J. S. (1975) Chem. Commun., 936-937]. The chemical shifts of the C-2 and C-4 protons of histidine-12 of S-peptide are followed as a function of pH and a pK' value of 6.75 is obtained. The reassignment of the three C-2 histidine resonances of S-protein is confirmed by partial deuteration studies. The pK' values obtained from titration of the H-2 resonances of His-48, His-105 and His-119 are 5.3, 6.5 and 6.0, respectively. The S-protein is less stable to acid than RNase-A since the former, but not the latter, shows evidence of reversible denaturation at pH 3 and 26 degrees C. His-48 in S-protein titrates normally and has a lower pK than in RN-ase-A probably because of the absence of Asp-14, which in RN-ase-A forms a a hydrogen bond with His-48 and causes it to be inaccessible to solvent, at pH values below 9.