A study of the histidine residues of human carbonic anhydrase B using 270 MHz proton magnetic resonance

Nine resonances in the 270 MHz proton magnetic resonance spectrum of human carbonic anhydrase B have been identified with imidazole C₍₂₎ protons of histidine residues, six of which are observed to titrate with pK_a values in the range 4.7 to 7.4. The behaviour of the nine resonances has been studied in the presence of the inhibitors, iodide, cyanide, acetate, hexacyanochromate, and imidazole. Measurements have also been made of the enzyme in its apo, cobalt, and mono-alkylated forms. Used in conjunction with the crystal structure, these results have enabled the tentative assignment of all nine resonances to particular histidine residues in the amino-acid sequence. Three of the active-site histidines at positions 64, 67, and 200 have low pK_a values and cannot be directly linked to the activity of the enzyme. However, the resonances assigned to the three metal-liganding histidines do exhibit changes on anion binding and with pH, which parallel changes in the esterase activity. These results are consistent with the model of an ionizable water molecule bound to the zinc ion.Linewidth measurements of the resonances of the histidine residues on the enzyme surface are used to estimate pseudo-first-order rate constants of the order of 4 × 10³ s^?1 for D⁺ exchange between imidazole N and solvent in the absence of buffer. These rates are observed to increase in the presence of small amounts of the buffers Tris and imidazole.     

Studies of the histidine residues of human and bovine glycoprotein hormones by nuclear magnetic resonance

Titration curves of the histidine residues in lutropin, thyrotropin, follitropin and chorionic gonadotropin have been assigned using imidazole C-2 proton nuclear magnetic resonance spectra and their estimated pK values determined. Spectra of reassociated hormone preparations, in which one or the other of their two subunits (alpha or beta) have had their accessible histidines exchanged with deuterium, permitted assignment of C-2 resonance to specific residues. Similar titration curves were found for residues which are conserved from one hormone to another. However, these conserved histidines do not have identical pK values, indicating that differences in the conformation or microenvironment around these residues occur in these hormones. Changes in some pK values also occur as a function of subunit association. The most dramatic change seen in all cases is the exposure to solvent of histidine alpha-83; in isolated alpha subunits this residue is unavailable for titration over a wide pH range. This change appears to be a general consequence of the association of the two subunits in any of these hormones. The data show that all histidines in the intact hormones are accessible to the environment, including those proposed to be in domains involved in subunit-subunit interaction.     

1H nuclear magnetic resonance study of the histidine residues of insulin

Insulin has proved difficult to study by nuclear magnetic resonance spectroscopy because of its complex aggregation behaviour in solution and its insolubility between pH 4 and 7. Now for the first time it has been possible to assign the ¹H nuclear magnetic resonances of the H-2 histidine protons of residues B5 and B10 of bovine 2 Zn insulin and Zn-free insulin, and the B5 and A8 residues of hagfish insulin. As expected, the addition of Zn to Zn-free insulin causes virtually no change in the chemical shift or the rate of H-D exchange of the H-2 proton of histidine B5, which is not involved in Zn binding in the 2 Zn insulin hexamer. The rate of H-D exchange of the H-2 proton of histidine B10 is decreased markedly on Zn binding at this residue, but the chemical shift of the resonance remains virtually constant owing to the balancing of an upfield ring current shift of the ordered histidine residues by a downfield shift due to electron withdrawal from the ring nitrogen by the Zn binding.     

Magnetic resonance study of exchangeable protons in human carbonic anhydrases.

A titratable exchangeable proton resonance assignable to a histidine imidazole ring N--H proton is observed approximately minus 15 ppm downfield from tetramethylsilane. The chemical shift of this resonance is affected by sulfonamide and anion inhibitors, and by removal of zinc or replacement of zinc by cobalt, indicating that the proton is located at or near the active site. The pH dependence of the chemical shift of this resonance, which is abolished by inhibitors, reflects the titration of a group with a pK-a of 7.3 in human carbonic anhydrase B and smaller than or equal to 7.1 in human carbonic anhydrase C. These pK-a values are interpreted to be due to the ionization of a neutral imidazole to form the imidazolate anion coordinated to zinc. A mechanism for enzymatic catalysis involving reversible deprotonation and coordination of a histidine to the metal is consistent with these studies.     

Proton magnetic resonance studies of histidines in human, rhesus monkey, and bovine carbonic anhydrases.

Histidine C-2 proton resonances in rhesus monkey carbonic anhydrase B (carbonate hydro-lyase, EC 4.2.1.1) and bovine carbonic anhydrase were investigated using 270-MHz proton magnetic resonance. The results suggest that there are extensive three-dimensional homologies between the human B and rhesus B enzymes and between the human C and bovine enzymes. Resonances from solvent exchangeable protons have been observed in the 11-16 ppm range in the NMR spectra of human carbonic anhydrases B and C and bovine carbonic anhydrase. Up to five of these are sensitive to changes of pH and the presence of inhibitors. Three of these resonances are assigned to NH protons of the metal coordinated imidazole groups. These results are discussed in relation to various models for the catalytic mechanism of carbonic anhydrase.     

Direct proton magnetic resonance determination of the pKa of the active center histidine in thiolsubtilisin

The serine proteases constitute a group of endopeptidases whose members owe their catalytic activity to the presence of a catalytic triad of amino acids consisting of a serine, a histidine and an aspartate. The pK(a) values for this histidine have been determined for several cases in which there is a negative charge installed at the serine to mimic the oxyanionic intermediate and related transition state for the catalytic pathway. Instances from this laboratory include (1) replacement of the serine by a cysteine in subtilisin to create a thiolate; (2) formation of monoisopropylphosphoryl-Ser 195 monoanionic phosphodiesters (in trypsin and chymotrypsin, Ser 221 in subtilisins); and (3) tetrahedral boronates formed with peptide boronic acids. The nuclear magnetic resonance (NMR) signals pertinent to this histidine, or signals indirectly reflecting the state of ionization of this histidine, have been used effectively to monitor changes in the active center ionization state. In every case studied, there is elevation of the pK(a) at the histidine when the negative charge is installed at the serine position. Herein is reported the first NMR measurement of the active center His 63 pK(a) in thiolsubtilisin Carlsberg; it is elevated by 3 units compared with the parent enzyme. Using a numerical solution (finite difference) of the Poisson-Boltzmann equation, a protein dielectric constant of 4 provides a good estimate of the experimentally observed pK(a) elevations. Very significantly, a very low protein dielectric constant (epsilon(p) = 3-5) is required in all of the comparisons, and for all three enzymes used (chymotrypsin, trypsin, and subtilisin). Finally, we discuss why the electrostatic perturbation sensed at His of the active center is more amplified by a negative charge on the Ser side than the same charge on the Asp side. A plausible explanation is that the positive charge on the imidazolium ring of the His is localized, with the N(delta 1) carrying a smaller fraction, the N(epsilon 2) carrying the bulk of the positive charge.     

Catalytic mechanism of alpha-class carbonic anhydrases: CO2 hydration and proton transfer

Carbonic anhydrases were first identified in red blood cells and have been thus traditionally addressed in a hematological context. However, recently there has been a shift of research interest to therapeutic areas, notably in solid cancers, relegating the impact of carbonic anhydrase function and pathological dysfunction in blood related physiology to secondary importance. This review addresses this paradigm and emphasizes the potential impact of recent studies on blood related carbonic anhydrase isotype expression and modulation in diverse areas such as physiology and pathology, biosensing, their use as biomarkers, and in the development of synthetic blood. A special emphasis is placed on reviewing new dynamic and quantitative studies that allow for the efficient tracking and quantitation of various carbonic anhydrase isozymes within the blood and more generally within the human body, that give new perspectives on the biochemical and physiological role of blood associated carbonic anhydrase in health and pathology.     

Proton magnetic resonance studies of carbonic anhydrase. I. Identification of histidine resonances.

Nuclear magnetic resonance (nmr) spectra of human carbonic anhydrase B recorded in deuterium oxide reveal seven discrete single proton resonances between 7 and 9 ppm downfield from sodium 2,2-dimethyl-i-silapentane-5-sulfonate. Simplification of spectra by use of Fremy's salt, comparison of peak widths at intersections, and evaluation of the results of inhibition and modification experiments permit determination of the pH dependencies of these resonances. Five of these peaks change position with increasing pH; three move upfield by approximately 95 Hz and two move downfield by 10 and 23 Hz. The first three reflect residues with pK values of 7.23, 6.98, and 6 and can be assigned to the C-2 protons of histidines. The two remaining pH dependent resonances reflect groups with pK values of 8.2 and 8.24. Their line widths and T1 values are comparable to those of the first group, and they also appear to reflect C-H protons of histidines. Despite the structural and functional similarities of the B and C isozymes of human carbonic anhydrase, few of the low field resonances appear to be common to both. Six histidine C-2 protons are observed in the C enzyme and reflect groups with pK values of approximately 7.3, 6.5, 5.7, 6.6, 6.6, and 6.4. A seventh peak contains two protons and moves upfield with increasing pH without titrating. A final resonance to low field moves downfield with increasing pH and reflects a group with a pK between 6 and 7. Its behavior resembles that of peak 1 of the human B enzyme, and it also appears to be a histidine C-H proton. This peak may reflect a conserved residue in the two isozymes that plays an important role in enzymatic function, as discussed in the following paper.     

Chemical rescue of proton transfer in catalysis by carbonic anhydrases in the beta- and gamma-class

Catalysis of the dehydration of HCO(3)(-) by carbonic anhydrase requires proton transfer from solution to the zinc-bound hydroxide. Carbonic anhydrases in each of the alpha, beta, and gamma classes, examples of convergent evolution, appear to have a side chain extending into the active site cavity that acts as a proton shuttle to facilitate this proton transfer, with His 64 being the most prominent example in the alpha class. We have investigated chemical rescue of mutants in two of these classes in which a proton shuttle has been replaced with a residue that does not transfer protons: H216N carbonic anhydrase from Arabidopsis thaliana (beta class) and E84A carbonic anhydrase from the archeon Methanosarcina thermophila (gamma class). A series of structurally homologous imidazole and pyridine buffers were used as proton acceptors in the activation of CO(2) hydration at steady state and as proton donors of the exchange of (18)O between CO(2) and water at chemical equilibrium. Free energy plots of the rate constants for this intermolecular proton transfer as a function of the difference in pK(a) of donor and acceptor showed extensive curvature, indicating a small intrinsic kinetic barrier for the proton transfers. Application of Marcus rate theory allowed quantitative estimates of the intrinsic kinetic barrier which were near 0.3 kcal/mol with work functions in the range of 7-11 kcal/mol for mutants in the beta and gamma class, similar to results obtained for mutants of carbonic anhydrase in the alpha class. The low values of the intrinsic kinetic barrier for all three classes of carbonic anhydrase reflect proton transfer processes that are consistent with a model of very rapid proton transfer through a flexible matrix of hydrogen-bonded solvent structures sequestered within the active sites of the carbonic anhydrases.     

In vivo targeting of tumor-associated carbonic anhydrases using acetazolamide derivatives

We describe the synthesis and characterization of two acetazolamide derivatives containing either a charged fluorophore or an albumin-binding moiety, which restrict binding to carbonic anhydrase IX and XII present on tumor cells. In vivo studies showed the preferentially targeting of tumor cells by the fluorescent acetazolamide derivative and the ability of the albumin-binding acetazolamide derivative to cause tumor retardation in a SK-RC-52 xenograft model of cancer.     

Large-scale preparation of the human carbonic anhydrases

A procedure for the large-scale preparation of human carbonic anhydrases B and C is described. The procedure has been adopted for routine use in this laboratory for preparing the large amounts of protein required for primary structural studies on both enzymes.