Mapping the Active Sites of 3-Phosphoglycerate Kinase and Glycerol Kinase with Monoammine Chromium(III) ATP

• Published in: Biochemistry (Easton) 1998-04, Vol.37 (16), p.5349-5355
• Main Authors: Lester, Lynn M, Rusch, Laura A, Robinson, Greg J, Speckhard, David C
• Format: Article
• Language: English
• Subjects: 3-phosphoglycerate kinase; Adenosine Diphosphate - chemistry; Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - chemistry; Adenosine Triphosphate - metabolism; Adenylyl Imidodiphosphate - chemistry; Binding Sites; Candida mycoderma; Chromium - chemistry; Glycerol Kinase - chemistry; Glycerol Kinase - metabolism; Hexokinase - chemistry; Isomerism; Kinetics; Models, Molecular; monoammine chromium(III) ATP; Organometallic Compounds - chemistry; Organometallic Compounds - metabolism; Phosphoglycerate Kinase - chemistry; Phosphoglycerate Kinase - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi980169d

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-phosphoglycerate kinase show a dramatic decrease in isomer binding only when the ammonia is in the Δ axial facial anti position. This suggests an open site architecture with only one strong contact point between the coordination sphere and the enzyme surface. These results agree well with the computer modeling studies of bidentate chromium ATP into the nucleotide site determined by X-ray crystallography [McPhillips, T., et al. (1996) Biochemistry 35, 4118−4127]. Both methods describe an open site strongly supporting the validity of the inhibition studies. Inhibition studies of glycerol kinase show significant decreases in binding for all the tested ammonia positions, suggesting a closed site architecture 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 glycerol kinase. Inhibition studies of hexokinase previously reported [Rawlings, J., et al. (1993) Biochemistry 32, 11204−11210] more closely resemble those of 3-phosphoglycerate kinase, suggesting the surprising result that however closely hexokinase and glycerol kinase are related structurally the site around the coordination sphere in hexokinase is functionally open like that of 3-phosphoglycerate kinase.