Hydrolysis of trifluoroethyl phosphate as evidence that the serine and tyrosine phosphatase activities of calcineurin share the same specificity determinant.
Calcineurin is shown to hydrolyze 2,2,2-trifluoroethyl phosphate. The time course of hydrolysis showed a lag period. The lag was present at all substrate concentrations used and was dependent on substrate concentration. Evaluation of the kinetic constants from the steady state portion of the enzymatic reaction indicated that hydrolysis of this compound fit the Bronsted relationship determined previously for Vmax/Km term for the hydrolysis of aromatic phosphate esters. Moreover, a Bronsted relationship for the Vmax was developed for the substrates containing leaving groups that are more basic than 2,3,4,5-tetrafluorotyrosine. The hydrolysis of 2,3,4,5-tetrafluorotyrosine phosphate did not fit this relationship implying a change in the rate limiting step with this substrate. The significance of these data to the serine and tyrosine phosphatase activities of calcineurin is discussed. A rationale for understanding the determinant of both activities of calcineurin is presented.
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
B L
BL
Martin
D J
DJ
Graves
D000818:Animals; D001921:Brain / Q000201:enzymology; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D006868:Hydrolysis; D007700:Kinetics; D010755:Organophosphates / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D013379:Substrate Specificity
D002148:Calmodulin-Binding Proteins; D010755:Organophosphates; C082183:2,2,2-trifluoroethyl phosphate; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases