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

Publisher Website
Google Scholar
Abstract
The preparations of [.beta..gamma.-18O,.gamma.-18O3]ATP and [.beta..gamma.-18O,.beta.-18O2]ATP and a proceudre for studying the positional isotopic exchange method of Midelfort and Rose 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 [Escherichia coli] 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.