Coenzyme Interactions: VI. PROPERTIES OF THE PYRIMIDINE MOIETY OF THIAMINE

Abstract

Abstract A discrete proton magnetic resonance signal is observed for the amino protons of the pyrimidine moiety of thiamine in water from pH 0 to pH 9; the integrated area of this signal is indicative of only two protons, confirming that the NH2 group is not the site of protonation of the aminopyrimidine moiety. The chemical shift of the (C-4') —NH2 signal is pH dependent, changing approximately 1.6 ppm over the range pH 0 to pH 7. A plot of chemical shift versus pH describes a curve with an inflection point at pH 5, corresponding to the pK1 for protonation of the pyrimidine moiety. The chemical shifts of all the other signals in the spectrum of thiamine are affected much less by pH changes, but all, like the (C-4')—NH2 signal, show a linear dependence on the fraction of thiamine in the protonated form. In addition, the line width of the amino signal is pH dependent, and a detailed study of changes in line width under various conditions suggest three mechanisms of exchange of the NH2 protons: (a) specific acidic catalysis, K30°h+ = 6.8 x 102 m-1 sec-1; (b) general acid-base catalysis, K30°thiamine = 1.8 x 102 m-1 sec-1; K30°acetate = 3.3 x 102 m-1 sec-1; (c) specific basic catalysis, K30°oh- = 1.6 x 108 m-1 sec -1. The energy of activation of the amino proton exchange was found to be similar for specific catalysis by H+ and OH-, and general acid-base catalysis by thiamine and by acetate; Ea = 12.5 to 12.9 Kcal per mole. The changes in width of the water signal were qualitatively similar to those of the thiamine (C-4')—NH2 signal. H-D exchange at the C-2 atom of the thiazolium ring proceeds approximately 10 times faster in the case of thiamine than in the case of oxythiamine, over the range of pH 3 to 6. The pK1 value for thiamine is lowered approximately 0.5 unit by complex formation with tryptophol in 50% methanol-water. Determination of apparent association constant for the 1:1 complex from potentiometric titration data (ΔpK) agreed with the value calculated from proton magnetic resonance data (Δδ), Kassoc = 1.5 m-1.