Measured change in protein solvation with substrate binding and turnover.
Osmotic stress is used to measure solvation changes that accompany the conformational changes of an active enzyme. For hexokinase both the equilibrium dissociation constant and the kinetic Michaelis-Menten constant for glucose vary linearly, and to the same extent, with the activity of water in the protein medium, as adjusted with large molecular weight (> 2000) osmolytes. The variation over the whole osmotic pressure range studied indicates that glucose binding is accompanied by the release of at least 65 +/- 10 water molecules, and this is reversed on enzyme turnover. The results indicate that near the physiological range of pressures the number may be higher. Most of this water, which behaves like an inhibitor, likely comes from the cleft which is induced to close around the substrate. Such large dehydration/rehydration reactions during turnover imply a significant contribution of solvation to the energetics of the conformational changes. Osmotic stress is a method of general applicability to probe water's contribution to functioning molecules.
Brock University, St. Catharines, Ontario, Canada.
R P
RP
Rand
N L
NL
Fuller
P
P
Butko
G
G
Francis
P
P
Nicholls
D002384:Catalysis; D005947:Glucose / Q000378:metabolism; D006593:Hexokinase / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D009997:Osmotic Pressure; D011092:Polyethylene Glycols / Q000737:chemistry; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D014867:Water / Q000737:chemistry
D011506:Proteins; D014867:Water; D011092:Polyethylene Glycols; D006593:Hexokinase; D005947:Glucose