Application of 19F nuclear magnetic resonance to examine covalent modification reactions of tyrosyl derivatives: a study of calcineurin catalysis.
The hydrolysis of fluorotyrosine phosphate by the calmodulin-activated phosphatase calcineurin has been monitored by 19F nuclear magnetic resonance spectroscopy. Previous work had established that the 19F nuclear magnetic resonance shift of the fluorine nucleus was altered after the phosphorylation of the phenolic hydroxyl group (B. Martin, C.J. Pallen, J.H. Wang, and D.J. Graves (1985) J. Biol. Chem. 260, 14592-14597). The disappearance of substrate and the appearance of product can be measured simultaneously with this approach. Application of the integrated form of the Michaelis-Menten equation yields estimates of the kinetic parameter, KM, close to the values obtained by initial rate kinetics. The velocity term, VM, was also evaluated to be approximately the same value. Calcineurin was determined not to be inactivated over the time period of the reaction. The results demonstrate that 19F nuclear magnetic resonance spectroscopy can be applied to the examination of enzyme-catalyzed reactions.
Martin
B L
BL
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Graves
D J
DJ
D000818:Animals; D001921:Brain / Q000201:enzymology; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002384:Catalysis; D002417:Cattle; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D002148:Calmodulin-Binding Proteins; C001245:3-fluorotyrosine; D014443:Tyrosine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases