A method for determining the positional isotope exchange in a nucleoside triphosphate: cyclization of nucleoside...

The preparations of [beta gamma-18O,gamma-18O3]ATP and [beta gamma-18O,beta-18O2]ATP and a procedure for studying the positional isotopic exchange method of Midelfort and Rose [Midelfort, C.F., & Rose, I.A. (1976) J. Biol. Chem. 251, 5881-5887] are described for detection of transient ATP cleavage to ADP in enzyme active sites. ATP is cyclized by reaction with dicyclohexylcarbodiimide in anhydrous pyridine to give adenosine 5'-trimetaphosphate. This rapidly undergoes ring opening on adding water to re-form ATP. This results in the randomization of the beta- and gamma-phosphates. The new gamma-phosphate is hydrolyzed to Pi by using glycerokinase and D-glyceraldehyde. Mass spectral analysis of the 18O distribution in this Pi allows the extent of positional isotopic exchange to be determined. Procedures suitable for use with GTP are also described.