Functional studies of rat galactokinase.
Galactokinase is an ATP-dependent enzyme that catalyzes the phosphorylation of galactose to form galactose-1-phosphate. The defect in human galactokinase can result in the disease of galactosemia. On the other hand, the control of galactose-1-phosphate production by inhibiting galactokinase is a potential therapy for another disease referred to as classic galactosemia. Many pharmaceutically important compounds derive from carbohydrate-containing natural products, and glycorandomization is one of the most efficient approaches for complex secondary metabolites. Therefore, it is important to further understand the interaction between galactokinase and its substrate or substrate analogs. In the present study, we cloned and purified both N- and C-terminal His-tagged rat galactokinase. We then constructed and purified a variety of variant enzymes, which were studied using kinetics with galactose and its analogs as substrates. We found that the binding of the ATP may induce conformational change to the enzyme so that the enzyme can bind galactose specifically. Asp186 was found to be a possible catalytic residue. The mutants were incubated with fluorescent trinitrophenyl-ATP for the characterization of their ATP binding sites. Various other substrate analogs, aminoglycosides and flavanoids were also tested and found to be competitive inhibitors of rat galactokinase.
Department of Biology and Chemistry, City University of Hong Kong, Hong Kong SAR, China.
Xiusheng
X
Chu
Nan
N
Li
Xiaojun
X
Liu
Ding
D
Li
D000255:Adenosine Triphosphate / Q000378:metabolism; D000617:Aminoglycosides / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D005419:Flavonoids / Q000378:metabolism; D005686:Galactokinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005690:Galactose / Q000031:analogs & derivatives / Q000378:metabolism*; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009154:Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D000617:Aminoglycosides; D005419:Flavonoids; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D005686:Galactokinase; D005690:Galactose