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

Biochemistry. 1980 Oct 14;19(21):4744-8. doi: 10.1021/bi00562a004.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate*
  • Carbodiimides*
  • Chemical Phenomena
  • Chemistry
  • Cyclization
  • Dicyclohexylcarbodiimide*
  • Guanosine Triphosphate*
  • Magnetic Resonance Spectroscopy
  • Mass Spectrometry
  • Oxygen Isotopes

Substances

  • Carbodiimides
  • Oxygen Isotopes
  • Dicyclohexylcarbodiimide
  • Guanosine Triphosphate
  • Adenosine Triphosphate