Providing a chemical basis toward understanding the histidine base-on motif of methylcobalamin-dependent methionine synthase: an improved purification of methylcobinamide, plus thermodynamic studies of methylcobinamide binding exogenous imidazole and pyridine bases.

Reported herein are the synthesis and improved purification of MeCbi(+).BF(4)(-) leading to 95% pure product. The availability of this higher purity MeCbi(+).BF(4)(-) has, in turn, allowed a study of the K(assoc), DeltaH, and DeltaS for exogenous imidazole and pyridine bases binding to MeCbi(+) in ethylene glycol and buffered aqueous solution. The results show that (1) the bases studied have larger K(assoc) values (where measurable) when binding to MeCbi(+) than when binding to AdoCbi(+) under analogous conditions; (2) comparison of the thermodynamic binding parameters for py and N-MeIm show that these bases bind similarly, within experimental error to MeCbi(+), contrary to what was seen earlier with AdoCbi(+); (3) the bases follow the expected trend, with the base with the highest pK(a) of those studied, 4-Me(2)Npy, exhibiting the highest K(assoc) value (K(assoc)(25 degrees C) = 18.0 +/- 0.3 M(-1)) and the base of lowest pK(a), py, exhibiting the lowest detectable K(assoc) value (K(assoc) (25 degrees C) = 6.2 +/- 0.4 M(-1)); (4) there is no detectable binding (K(assoc) = 0.07 M(-1)) for 2-Mepy or 2,6-Me(2)py with MeCbi(+); and (5) the base that is closest to the biologically relevant axial His759 residue in methionine synthase, N-MeIm, exhibits an unusual DeltaH value for the formation of MeCbi(+).N-MeIm, results interpreted as offering further support for the presence of sigma plus pi effects when imidazole bases bind to alkylcobinamides. The results of these studies allow the percentage of base-on methylcobinamide, MeCbi(+).base, to be calculated as a function of temperature and added base. As such, they provide necessary background information for RS(-) + MeCbi(+).base and other methionine synthase chemical precedent studies.