Determination of kinetic isotope effects for nucleoside hydrolases using gas chromatography/mass spectrometry

Kinetic isotope effects are widely used to determine the transition slate o f chemical and enzymatic reactions. Radioactive isotopes are used most ofte n to determine these kinetic isotope effects. However, stable isotopes offe r a number of advantages over the use of radioactive isotopes. These advant ages include ease of handling and disposal along with increased safety in t he laboratory. [1'-C-13]Inosine and [1'-H-2]inosine kinetic isotope effects were determined using a gas chromatograph in conjunction with a mass selec tive detector for nucleoside hydrolase, a purine-metabolizing enzyme. Three ion pairs were used to determine kinetic isotope effects. These ion pairs were 158/159, 187/188, and 217/218. The average isotope effects for all ion pairs were 1.021 +/- 0.006 for [1'-C-13]inosine and 1.113 +/- 0.008 for [1 '-H-2]inosine. The transition state consistent with these isotope effects i s also consistent with the transition state proposed by Schramm and Horenst ein using radioactive substrates. (C) 1999 Academic Press.