MAPPING THE ACTIVE-SITES OF 3-PHOSPHOGLYCERATE KINASE AND GLYCEROL KINASE WITH MONOAMMINE CHROMIUM(III) ATP

The 12 isomers of monoammine chromium(III) ATP have been used to probe the ATP binding sites of yeast 3-phosphoglycerate kinase and glycerol kinase from Candida mycoderma. Inhibition studies of 3-phosphoglycera te kinase show a dramatic decrease in isomer binding only when the amm onia is in the Delta axial facial anti position. This suggests an open site architecture with only one strong contact point between the coor dination sphere and the enzyme surface. These results agree well with the computer modeling studies of bidentate chromium ATP into the nucle otide site determined by X-ray crystallography [McPhillips, T., et al. (1996) Biochemistry 35, 4118-4127]. Both methods describe an open sit e strongly supporting the validity of the inhibition studies. Inhibiti on studies of glycerol kinase show significant decreases in binding fo r all the tested ammonia positions, suggesting a closed site architect ure with many contacts between the coordination sphere and the surface of the enzyme. This is in good agreement with X-ray studies [Hurley, T., et al, (1993) Science 259, 673-677] on the Escherichia coli glycer ol kinase. Inhibition studies of hexokinase previously reported [Rawli ngs, J., et al. (1993) Biochemistry 32, 11204-11210] more closely rese mble those of 3-phosphoglycerate kinase, suggesting the surprising res ult that however closely hexokinase and glycerol kinase are related st ructurally the site around the coordination sphere in hexokinase is fu nctionally open like that of 3-phosphoglycerate kinase.