Carbon-13 magnetic resonance of carboxymethylated human carbonic anhydrase B. Chemical shift and spin--lattice relaxation studies.

We have previously prepared Ntau-carbosymethylhistidine-200 human carbonic anhydrase B using 90% [1-13C]bromoacetate and have observed the 13C NMR resonance of the enriched carboxylate now covalently attached in the active site. We report here chemical shift studies of the zinc-free carboxymethylated enzyme and its Co2+-substituted form, as well as relaxation studies of the resonance in the zinc enzyme at three frequencies (15.04, 25.15, and 90.5 MHz). The chemical shift and relaxation data are both consistent with the immobilization of the carboxylate at pH 8 and its approach or coordination to the zinc. The relaxation data indicate that lowering the pH to 5.5 leads to internal motion of the carboxymethyl moiety, consistent with the chemical shift evidence for the disruption of the proposed zinc--carboxylate coordination. Inhibitor binding at either pH 5.5 or 8.0 eliminates whatever internal motion might be present. The relaxation data have been interpreted using theoretical calculations on dipolar and chemical shift anisotropy contributions. The combined results indicate that the catalytic consequences of the carboxymethylation may be due to the proposed zinc--carboxylate coordination and need not result from the disruption of any role that histidine-200 might play in the catalytic mechanism.     

Histidine-200 alters inhibitor binding in human carbonic anhydrase B. A carbon-13 nuclear magnetic resonance identification.

We have previously prepared 13C-enriched NT-carboxymethylhistidine-200 human carbonic anhydrase B (CmHCAB) by reacting the native enzyme with 90% [1-13C]bromoacetate (Strader, D.J., and Khalifah, R.G. (1976), J. Am. Chem. Soc. 98, 5043). The 13C nuclear magnetic resonance signal of the enriched carboxylate of CmHCAB proved sensitive to active-site events, permitting, among other things, the determination of the microscopic pKa of the modified histidine. This report extends the study to the complexes of CmHCAB with the inhibitors iodide and azide. It is found that the pKa of histidine-200 is significantly increased when these inhibitors bind. A quantiative comparison of the iodide-induced pKa shift with literature data (Whitney, P. L., and Brandt, H. (1976), J. Biol, Chem. 251, 3862) showing that the binding of iodide is influenced by the ionization of an active-site group of pKa 6.1 allowed the clear identification of histidine-200 as the perturbing group. Other important implications of the magnetic resonance results are also discussed.     

Carbon-13 nuclear magnetic resonance studies of cobalamins

The carbon-13 nuclear magnetic resonance spectra of aquocobalamin, adenosylcobalamin, methylcobalamin, and (carboxymethyl)cobalamin have been interpreted. The assignments were made by a comparison of the spectra with that of cyanocobalamin, by a study of the pH dependence of the chemical shifts, by an analysis of the effect of the axial ligands on the carbon atoms of the corrin ring, and by a study of the specific line broadening effect of the paramagnetic ions Mn2+ and Gd3+. The chemical shift changes that accompany the "base-on"----"base-off" conversion of the organocobalamins demonstrate that the conformation of the "western" half of the corrin ring and the conformations of the a, b, c, d, f, and g side chains are relatively constant. In contrast, the conformations of the "eastern" half of the corrin ring and the e propionamide side chain are highly variable.     

Carbon-13 nuclear magnetic resonance spectra of lignins

From the ¹³C-nmr spectra of a large number of dimeric and monomeric lignin model compounds the chemical shifts of the carbon atoms of the C₉-units in lignin with different substitution patterns were determined. The absorption peaks of the carbon-13 spectra of two lignins (beech and spruce) could be assigned by comparison (Table 3).     

Carbon-13 nuclear magnetic resonance spectral analysis of C-nucleosides. The structure of pyrazomycin B 1

The ¹³C nmr spectra of three nucleosides and four C-nucleosides have been recorded and all carbon signals assigned. These data have been utilized for the determination of the structure and conformation of the antibiotic pyrazomycin B. Steric differences have been shown to be reflected in the chemical shift values.     

Carbon-13 nuclear magnetic resonance spectra of carbohydrate oxirane derivatives

The ¹³C-chemical shifts and ¹J_C,H values of two series of carbohydrate oxirane derivatives, namely methyl 2,3-anhydro-ribo- and -lyxofuranosides and methyl 2,3-anhydro-4,6-O-benzylidene-manno- and -allopyranosides have been determined. The assignment of ¹³C resonances has been established mainly by the examination of the proton-coupled and the selective proton-decoupled spectra. The effect of the oxirane rings on the chemical shifts of β and γ carbon atoms (from the oxirane ring oxygen atom) has been observed. Large ¹J_C,H values associated with cis CH bonds adjacent to the oxirane rings relative to those of trans counterparts have been found.     

Metabolism of methanol in plant cells. Carbon-13 nuclear magnetic resonance studies

Using (13)C-NMR, we demonstrate that [(13)C]methanol readily entered sycamore (Acer pseudoplatanus L.) cells to be slowly metabolized to [3-(13)C]serine, [(13)CH(3)]methionine, and [(13)CH(3)]phosphatidylcholine. We conclude that the assimilation of [(13)C]methanol occurs through the formation of (13)CH(3)H(4)Pte-glutamate (Glu)(n) and S-adenosyl-methionine, because feeding plant cells with [3-(13)CH(3)]serine, the direct precursor of (13)CH(2)H(4)Pte-Glu(n), can perfectly mimic [(13)CH(3)]methanol for folate-mediated single-carbon metabolism. On the other hand, the metabolism of [(13)C]methanol in plant cells revealed assimilation of label into a new cellular product that was identified as [(13)CH(3)]methyl-beta-D-glucopyranoside. The de novo synthesis of methyl-beta-D-glucopyranoside induced by methanol did not require the formation of (13)CH(3)H(4)Pte-Glu(n) and was very likely catalyzed by a "transglycosylation" process.     

Proton nuclear magnetic resonance studies of histidines in horse carbonic anhydrase I

The 250 MHz 1H-NMR spectrum of horse carbonic anhydrase I (or B) (carbonate hydro-lyase, EC 4.2.1.1) was measured as a function of pH under various conditions. Eight resonances corresponding to histidine C-2 protons and four resonances corresponding to histidine C-4 protons were identified and assigned to individual histidine residues in the enzyme molecule. Substantial similarities between horse and human carbonic anhydrases I were demonstrated. While the human enzyme has three titratable histidine residues in its active site, the horse enzyme has only two, His-67 in the human enzyme being replaced by Gln in the horse enzyme (Jabusch, J.R., Bray, R.P. and Deutsch, H.F. (1980) J. Biol. Chem. 255, 9196-9204). This substitution has small but significant effects on the behaviour of the other active-site histidines. His-64 and His-200. However, His-64 has an anomalously low pKa value also in horse isoenzyme I, as previously observed in human isoenzyme I (Campbell, I.D., Lindskog, S. and White, A.I. (1974) J. Mol. Biol. 90, 469-489).     

A 13C nuclear magnetic resonance study of CO2/HCO-3 exchange catalyzed by human carbonic anhydrase I

Rates of CO2/HCO-3 exchange, catalyzed by human carbonic anhydrase I (or B) at chemical equilibrium, were estimated from the nuclear magnetic resonance linewidths of 13C-labeled substrates. The results show that the maximal exchange rate constant is independent of pH in the range 5.7-8.0, whereas the apparent substrate dissociation constant depends on pH. Exchange proceeds rapidly in the absence of added buffers, and the addition of buffers has negligible effects on exchange rates. Exchange is equally rapid with 1H2O or 2H2O as solvents. Chloride ions inhibit CO2/HCO-3 exchange competitively. The maximal exchange rates obtained with human carbonic anhydrase I are 50 times slower than those obtained with human isoenzyme II (or C). From a comparison of the exchange kinetics with the steady-state kinetics of CO2 hydration and HCO-3 dehydration it is tentatively concluded that the transfer of H+ between active site and medium proceeds with rates of similar magnitudes in the two isoenzymes, whereas the central catalytic step, the interconversion of enzyme-bound CO2 and HCO-3, is much slower in isoenzyme I than in isoenzyme II.     

Carbon-13 nuclear magnetic resonance spectroscopy of flavonoid and isoflavonoid compounds

The ¹³C NMR spectra of 15 flavonoid and 9 isoflavonoid substances of various ring C oxidiation states were analyzed and their carbon shifts assigned. In the case of 3 terpenic flavones and two glycoflavones linewidths were related qualitatively to molecular segmental motion.     

Carbon-13 nuclear magnetic resonance spectroscopy of [2-13C]carboxymethylcytochrome c.

Horse heart cytochrome c has been carboxymethylated under various reaction conditions using [2-13C]bromoacetate. Direct analysis of reaction products using 13C nuclear magnetic resonance spectroscopy shows that the protein can be much more extensively modified than has previously been assumed. The proximity of one carboxymethylmethionine residue to the paramagnetic center of the ferric protein allows it to be distinguished from a more constant carboxymethylmethionine residue on the basis of the chemical shift of its labeled methylene group. Refolding of cytochrome c after alkylation at low pH apparently gives a different configuration of modified methionine residues within the protein compared to that produced by alkylation at neutral pH in the presence of cyanide.     

1H nuclear magnetic resonance investigation of cobalt(II) substituted carbonic anhydrase.

The structure of ClO4 and NO3 adducts of cobalt(II) substituted bovine carbonic anhydrase have been investigated through 1D NOE and 2D 1H nuclear magnetic resonance (NMR) spectroscopy. For the first time two-dimensional NMR techniques are applied to paramagnetic metalloproteins other than iron-containing proteins. Several active site signals have been assigned to specific protons on the grounds of their scalar and dipolar connectivities and T1 values. The experimental dipolar shifts for the protons belonging to noncoordinated residues have allowed the identification of a plausible orientation of the magnetic susceptibility tensor around the cobalt ion as well as of the magnitude and the anisotropy of the principal susceptibility values. In turn, a few more signals have been tentatively assigned on the grounds of their predicted dipolar shifts. The two inhibitor derivatives have a very similar orientation but a different magnitude of the chi tensor, and the protein structure around the active site is highly maintained. The results encourage a more extensive use of the two-dimensional techniques for obtaining selective structural information on the active site of metalloenzymes. With this information at hand, comparisons within homologous series of adducts with various inhibitors and/or mutants of the same enzyme of known structure should be possible using limited sets of NMR data.     

Some properties of site-specific mutants of human carbonic anhydrase II having active-site residues characterizing carbonic anhydrase III

Four amino acid residues, His64, Asn67, Leu198 and Val207, in the active site of human carbonic anhydrase II, have been replaced by Lys64, Arg67, Phe198 and Ile207, which are characteristic for the muscle-specific, low-activity isoenzyme form, carbonic anhydrase III. The aim of the investigation has been to test if any of these residues, or a combination of them, is important for the low CO2 hydration activity, low esterase activity, low pKa for the pH/rate profile and low affinity for sulfonamide inhibitors characterizing carbonic anhydrases III. However, no evidence for such critical roles was found. A combination of Lys64 and Arg67 appears to result in a decrease in CO2 hydration activity, but even the quadruple mutant having all four changes is only eight times less active (kcat/Km) than unmodified isoenzyme II, in contrast to isoenzyme III which is nearly 300 times less active than isoenzyme II. The 4-nitrophenyl acetate hydrolase activity of the quadruple mutant is sevenfold lower than that of unmodified isoenzyme II, while the active site of isoenzyme III hardly catalyzes the hydrolysis of this ester at all. The pKa controlling the esterase activity of the quadruple mutant is 6.2, which should be compared to a value of 6.8 for unmodified isoenzyme II, and about 5 for isoenzyme III. While isoenzyme III binds sulfonamide inhibitors 10(3)-10(4) times less strongly than isoenzyme II, only [Asn-67----Arg]isoenzyme II shows a weaker binding of the investigated sulfonamide, dansylamide, but only by a factor of two. Some of the other mutants show enhanced affinities, up to nearly fourfold for the double mutant with Phe198 and Ile207. It is speculated that additional differences between the active sites of isoenzyme II and III might be important for the precise orientations and interactions of the side chains of isoenzyme-III-specific amino acid residues.     

Carbon-13 nuclear magnetic resonance studies on tobacco mosaic virus and its protein.

Fourier transform nuclear magnetic resonance studies on 12% 13C-enriched tobacco mosaic virus (TMV) and its rod-like protein oligomers in solution with molecular weights up to 42 X 10(6) are reported. In the virus approximately 17% of the carbons of the protein subunit have line widths of less than or equal to 300 Hz and T1 less than or equal to 1 s and are concluded to be mobile with more than one degree of freedom of internal rotation about a carbon--carbon bond. In the rodlike polymer of TMV protein at pH 5.3, 30% of the carbons are mobile, which implies rotational motions about carbon--carbon bonds and/or motions of the protein subunits within the polymer. The presence of internal mobility is supported by the observation that 20% of the carbons in the double disklike oligomer show decreasing line width upon increasing temperature; the remaining resonances have line widths which are temperature independent during the double disklike polymerization process. Since the molecular weight of TMV protein polymers increases with increasing temperature, this demonstrates that all nuclei within the double dislike oligomer are mobile. NMR and X-ray data on the double disklike polymer reveal differences with respect to internal mobility.     

Carbon-13 nuclear magnetic resonance spectra of D-homoannulated 17-hydroxypregnan-20-ones

The 13C-NMR spectra of several groups of isomeric D-homoannulated 17 alpha-hydroxypregnan-20-ones have been recorded. The chemical shifts of the various carbon atoms have been correlated with the structures of the different isomers.     

Carbon-13 nuclear magnetic resonance studies of spironolactone and several related steroids

The ¹³c chemical shifts for all the carbon atoms in spironolactone have been assigned. Assignments for nine additional steroids which include the C-7β isomer of spironolactone, its C-7 thiol hydrolysis product, the 7α-thioacetate derivative of testosterone and its thiol hydrolysis product are also reported.     

Carbon-13 nuclear magnetic resonance studies of adenosylcobalamin and alkylcorrinoids, selectively enriched with carbon-13.

The carbon-13 nuclear magnetic resonance spectra of a series of alkylcorrinoids, selectively enriched with 13C in the alkyl ligand, were recorded at 25.2 MHz and 25 degrees. The nature of the axial ligands markedly affects the chemical shift of the labeled alkyl moiety (trans effect) as well as the 13C resonances of selected carbon atoms of the corrin ring (cis effect). Although a number of factors appear to influence the trans effect on the chemical shift of the alkyl ligand (important among them being electric field effects), the cis effect appears to be dominated by changes in charge density (at the methine bridge carbon atoms, C-5, C-10, C-15) and by steric effects (at the methyl groups at C-1, C-5, and C-15) accompanying axial ligation. Spin-latice relaxation times of several organocorrinoids, selectively labeled with 13C in the ligands attached to cobalt, were also measured. The T1 values of the methylene carbons of [5'-13C]adenosylcobalamin and [2-13C]carboxymethylcobalamin are very similar to that of the methine bridge carbon atom C-10 of the corrin ring, indicating that rotation about the carbon-cobalt bond of these two corrinoids is severely restricted. On the other hand, internal rotation about the carbon-cobalt bond of methylcobalamin is rapid.     

Carbon-13 and proton nuclear magnetic resonance of unsonicated model and mitochondrial membranes

Proton nuclear magnetic resonance (PMR) spectra at 270 MHz of aqueous dispersions of nonsonicated egg lecithin, dipalmitoyl lecithin, egg lecithin-cholesterol (1 : 1) and dipalmitoyl lecithin-cholesterol (1 : 1), together with PMR spectra of mitochondrial membranes and their extracted lipids, have been obtained.Carbon-13 nuclear magnetic resonance (CMR) spectra at 25.2 MHz of egg lecithin, egg lecithin-cholesterol (1 : 1) and sphingomyelin, together with CMR spectra of chloroplast and mitochondrial membranes, and erythrocyte ghosts, have also been obtained. The results obtained using CMR appear very promising for further study of intact membrane structure.It is suggested, on the basis of CMR evidence, that the proteins in mitochondrial membranes may be considerably less mobile than the lipids.