aquacobalamin and cobamamide

Crystal structure analyses have helped to decipher the mode of binding of coenzyme B12 (AdoCbl) in the active site of AdoCbl-dependent enzymes. However, the question of how such enzymes perform their radical reactions is still incompletely answered. A pioneering study by Gruber and Kratky of AdoCbl-dependent glutamate mutase (GLM) laid out a path for the movement of the catalytically active 5'-deoxyadenosyl radical, in which H-bonds between the protein and the 2'- and 3'-OH groups of the protein bound AdoCbl would play a decisive role. Studies with correspondingly modified coenzyme B12-analogues are of interest to gain insights into cofactor binding and enzyme mechanism. Here we report the preparation of Coβ-2'-fluoro-2',5'-dideoxyadenosylcobalamin (2'FAdoCbl), which lacks the 2'-OH group critical for the interaction in enzymes. 2'FAdoCbl was prepared by alkylation of cob(I)alamin, obtained from the electrochemical reduction of aquocobalamin. Spectroscopic data and a single crystal X-ray analysis of 2'FAdoCbl established its structure, which was very similar to that one of coenzyme B12. 2'FAdoCbl is a (19)F NMR active mimic of coenzyme B12 that may help to gain insights into binding interactions of coenzyme B12 with AdoCbl-dependent enzymes, proteins of B12 transport and of AdoCbl-biosynthesis, as well as with B12-riboswitches.

Topics: Circular Dichroism; Cobamides; Crystallography, X-Ray; Fluorine; Magnetic Resonance Spectroscopy; Models, Chemical; Molecular Structure; Solutions; Vitamin B 12

Euglena aquacobalamin reductase (NADPH: EC 1.6.99.-) was purified, and its subcellular distribution was studied to elucidate the mechanism of the conversion of hydroxocobalamin to 5'-deoxyadenosylcobalamin. The enzyme was found in the mitochondria. It was purified about 150-fold over the Euglena mitochondrial extract in a yield of 38%. The purified enzyme was homogeneous in polyacrylamide gel electrophoresis. Spectra of the purified enzyme showed that it was a flavoprotein. The molecular weight of the enzyme was calculated to be 66,000 by Sephadex G-100 gel filtration and 65,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was specific to NADPH with an apparent Km of 43 microM and to hydroxocobalamin with an apparent Km of 55 microM. The enzyme did not require FAD or FMN as a cofactor. The optimum pH and temperature were 7.0 and 40 degrees C, respectively.

Topics: Animals; Chromatography, Gel; Cobamides; Electrophoresis, Polyacrylamide Gel; Euglena gracilis; Hydrogen-Ion Concentration; Hydroxocobalamin; Kinetics; Molecular Weight; NADH, NADPH Oxidoreductases; NADP; Temperature; Vitamin B 12

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.

Topics: Carbon; Chemical Phenomena; Chemistry; Cobamides; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Manganese; Molecular Conformation; Nucleosides; Nucleotides; Vitamin B 12