Steady-state carbon-13 nuclear magnetic resonance spectra of acyl-alpha-chymotrypsin

When [13C]carbonyl-enriched p-nitrophenyl 5-n-propyl-2-furoate is incubated with alpha-chymotrypsin, a new peak appears in the 13C NMR spectrum. On the basis of its position and the fact that it is "chased" with unlabeled substrate, we conclude that this new signal is due to the acyl-enzyme intermediate. In spectra taken during steady-state turnover, the acyl-enzyme ester carbonyl 13C chemical shift displays a pH dependence that fits to a titration curve with an apparent pK of 7.1 (0.1). The apparent pK of the kcat vs. pH curve for enzyme-catalyzed hydrolysis of the same substrate under conditions differing only in reactant concentration is 7.0 (0.1). We have found no spectral evidence for a tetrahedral intermediate.     

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).     

Proton and carbon-13 nuclear magnetic resonance studies of rhodopsin-phospholipid interactions

Proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR) spectra of rhodopsin-phospholipid membrane vesicles and sonicated disk membranes are presented and discussed. The presence of rhodopsin in egg phosphatidylcholine vesicles results in homogeneous broadening of the methylene and methyl resonances. This effect is enhanced with increasing rhodopsin content and decreased by increasing temperature. The proton NMR data indicate the phospholipid molecules exchange rapidly (less than 10(-3) s) between the bulk membrane lipid and the lipid in the immediate proximity of the rhodopsin. These interactions result in a reduction in either or both the frequency and amplitude of the tilting motion of the acyl chains. The 13C NMR spectra identify the acyl chains and the glycerol backbone as the major sites of protein lipid interaction. In the disk membranes the saturated sn-1 acyl chain is significantly more strongly immobilized than the polyunsaturated sn-2 acyl chain. This suggest a membrane model in which the lipid molecules preferentially solvate the protein with the sn-1 chain, which we term an edge-on orientation. The NMR data on rhodopsin-asolectin membrane vesicles demonstrate that the lipid composition is not altered during reconstitution of the membranes from purified rhodopsin and lipids in detergent.     

The fermentation of xylose: studies by carbon-13 nuclear magnetic resonance spectroscopy

Summary The fermentation ofd-xylose byPachysolen tannophilus, Candida shehatae, andPichia stipitis has been investigated by¹³C-nuclear magnetic resonance spectroscopy of both whole cells and extracts. The spectra of whole cells metabolizingd-xylose with natural isotopic abundance had significant resonance signals corresponding only to xylitol, ethanol and xylose. The spectra of whole cells in the presence of [1-¹³C]xylose or [2-¹³C]xylose had resonance signals corresponding to the C-1 or C-2, respectively, of xylose, the C-1 or C-2, respectively, of xylitol, and the C-2 or C-1, respectively, of ethanol. Xylitol was metabolized only in the presence of an electron acceptor (acetone) and the only identifiable product was ethanol. The fact that the amount of ethanol was insufficient to account for the xylitol metabolized indicates that an additional fate of xylitol carbon must exist, probably carbon dioxide. The rapid metabolism of xylulose to ethanol, xylitol and arabinitol indicates that xylulose is a true intermediate and that xylitol dehydrogenase catalyzes the reduction (or oxidation) with different stereochemical specificity from that which interconverts xylitol andd-xylulose. The amino acidl-alanine was identified by the resonance position of the C-3 carbon and by enzymatic analysis of incubation mixtures containing yeast and [1-¹³C]xylose or [1-¹³C]glucose. The position of the label from both substrates and the identification of isotope also in C-1 of alamine indicates flux through the transketolase/transaldolase pathway in the metabolism. The identification of a resonance signal corresponding to the C-1 of ethanol in spectra of yeast in the presence of [1-¹³C]xylose and fluoroacetate (but not arsenite) indicates the existence of equilibration of some precursor of ethanol (e.g. pyruvate) with a symmetric intermediate (e.g. fumarate or succinate) under these conditions.     

Tautomeric composition of D-fructose phosphates in solution by fourier transform carbon-13 nuclear magnetic resonance

The Fourier transform ¹³C magnetic resonance spectra of D-fructose 6-phosphate (F6P) and D-fructose 1,6-diphosphate (FDP) were obtained. The signal assignments made on the basis of ¹³C chemical shifts and ¹³C-³¹P spin-spin couplings indicate that the earlier assignments of the C-4 and C-5 resonances of α- and β-fructofuranose in oligosaccharides and D-fructose [Allerhand, A. and Doddrell, D., J. Amer. Chem. Soc., $\text{93}$, 2777, 2779 (1971)] should be reversed. Integration of signal intensities yields the following equilibrium composition at 35°C: F6P, α-anomer 19±2% and β-anomer 81±2%, FDP, α-anomer 23±4% and β-anomer 77±4%. Less than 1.5% keto or hydrated keto form is present in solutions of either fructose phosphate. The bearing of these findings on the tautomeric specificity of phosphofructokinase is discussed.     

Synthesis and carbon-13 nuclear magnetic resonance spectra of Cyclo(D-leucyl-L-leucine) and Cyclo(L-leucyl-L-leucine)

Cyclo(D -Leu-L -Leu) and cyclo(L -Leu-L -Leu) were synthesized, and their carbon-13 nmr spectra at 65 MHz were examined in dimethylsulfoxide and trifluoroacetic acid solutions. The chemical shift data are consistent with a boat or “twisted” boat conformation of the diketopiperazine ring in both solvents. There was no indication of protonation of the cyclic dipeptides by trifluoroacetic acid. Attempts at polymerizing the cyclic dipeptides were unsuccessful.     

FT-C13 nuclear magnetic resonance spectra of natural humic substances

The C13 resonance spectra of two humic acids extracted from Vertisol and Andosol soil and of a fulvic acid form Podzol soil are reported. The spectra were taken in 5 % W/W solution of the substances in 0.1 N NaOD in D₂O using the Fourier-transform-technique.     

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.     

Structural and conformational analysis of sialyloligosaccharides using carbon-13 nuclear magnetic resonance spectroscopy

The analysis of the carbon-13 chemical shift data of NeuAc alpha (2----3)Gal beta (1----4)Glc and NeuAc alpha (2----3)Gla beta-(1----4)GlcNAc and their respective NeuAc alpha (2----6) isomers established distinct and different conformations of the sialic acid residue, depending on the type of anomeric linkage [alpha-(2----3) vs. alpha (2----6)]. Interactions between the NeuAc residue and the Glc or GlcNAc residue are particularly strong in the case of the alpha (2----6) isomers. Similar effects are observed for the larger oligosaccharides [II3(NeuAc)2Lac and IV6NeuAcLcOse4] and even in intact glycoproteins and polysaccharides. It is proposed that the NeuAc alpha (2----3) isomers assume an extended conformation with the sialic residue at the end (terminal) of the oligosaccharide chain or branch. The NeuAc alpha (2----6) isomers are assumed to be folded back toward the inner core sugar residues.     

Structure and metabolism of mammalian liver glycogen monitored by carbon-13 nuclear magnetic resonance

Natural-abundance 13C NMR signals from glycogen are observable in situ within the perfused livers of rats. The nuclear magnetic relaxation properties (T1, T2, eta + 1) of glycogen were measured for glycogen in situ and in vitro and were found to be identical. All of the carbon nuclei in glycogen contribute to the high-resolution NMR spectrum, in spite of glycogen's very large molecular weight. The metabolism of glycogen in situ in the perfused rat liver was followed by 13C NMR. Stimulation of the fed rat liver by physiological glucagon levels led to rapid glycogenolysis. Perfusion of the liver with [1-13C]glucose led to net glycolysis, with concomitant scrambling of the label from C1 to C6 due to triosephosphate isomerase activity.     

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.     

Interactions of long-chain aldehydes with luciferase. A carbon-13 nuclear magnetic resonance study.

The interaction of long-chain aldehydes with bacterial luciferase has been studied by 13C NMR spectroscopy of natural-abundance and 13C-enriched 1-dodecanal. At high substrate/enzyme ratios, the spin-spin relaxation rates of C(1)-C(3) are faster than for the other carbons and are in the order C(1) greater than C(2) greater than C(3). The aldehyde is strongly bound in the active site along the entire length of the alkyl chain with the strongest interaction at the CHO group. At low substrate/enzyme ratios, interactions are apparent at C(10), which are removed upon denaturation of the enzyme. Spin-spin and spin-lattice relaxation rates were measured for odd-carbon 13C-enriched 1-dodecanal in the presence of luciferase. From the ratios of T1/T2 a single value of (1.8 +/- 0.7) X 10(-8) s was calculated for the rotational correlation time tc for the complex.     

Nuclear magnetic resonance studies on pyridine dinucleotides. The pH dependence of the carbon-13 nuclear magnetic resonance of NAD+ analogs.

The pH dependence of the ¹³C chemical shifts for nicotinamide adenine dinucleotide (NAD⁺), thionicotinamide adenine dinucleotide (TNAD⁺), pyridine adenine dinucleotide (PyrAD⁺), N-methyl-nicotinamide adenine dinucleotide (N-Me-NAD⁺), acetylpyridine adenine dinucleotide (AcPyAD⁺), nicotinamide hypoxanthine dinucleotide (NHD⁺), and nicotinamide adenine dinucleotide phosphate (NADP⁺) are reported. In these analogs the ¹³C chemical shifts of the pyridinium moiety reflect the pK_a of the opposing purine base, while the ¹³C chemical shift dependence on pD for the pyridinium carbons of nicotinamide mononucleotide (NMN⁺) and adenosine monophosphate (AMP), 1,4-dihydronicotinamide adenine dinucleotide (NADH), 1,4-dihydronicotinamide adenine dinucleotide phosphate (NADPH), and nicotinic acid adenine dinucleotide (N(a)AD⁺) are not influenced by the adenine ring in the pD range tested. Through the use of ¹³C-labeled NAD⁺, the source of the pH dependence of the ¹³C chemical shifts was shown to be intramolecular in origin. However, serious doubt is cast on the utility of employing the pD dependence of chemical shift data to determine the nature of solution conformers or their relative populations.