[Purification and mechanism of action of a plant galactokinase].

The previously described galactokinase from Fenugreek seeds, has been purified by affinity chromatography on a column of galactosamine-CH Sepharose. This material ensures a more specific fixation than does ATP-Sepharose. A 400 fold purification was achieved in a single step, with a 80 per cent yield. Km's for galactose and for Mg/ATP2- complex were respectively 0.54 x 10-3 M and 5, 10-3 M. Galactose-1-phosphate is a competitive inhibitor of galactose while the inhibition for Mg-ATP2- is not a competitive one. The Mg-ADP complex is a non-competitive inhibitor of both galactose and Mg-ATP2-. Moreover, the Km of the enzyme for M-ATP2- complex is modified when 2-deoxy- and 6-deoxy-galactose are used instead of galactose. These results are consistent with an ordered sequential mechanism for this galactokinase: galactose binds to the enzyme before Mg-ATP2-, and galactose-1-phosphate is the last reaction product liberated. The affinity of the kinase for 6-deoxygalactose is lower than for 2-deoxygalactose. This observation reveals the importance of the hydroxyl in C6 position for the binding on the enzyme.