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

Kinetic isotope effects are widely used to determine the transition state of chemical and enzymatic reactions. Radioactive isotopes are used most often to determine these kinetic isotope effects. However, stable isotopes offer a number of advantages over the use of radioactive isotopes. These advantages include ease of handling and disposal along with increased safety in the laboratory. [1'-(13)C]Inosine and [1'-(2)H]inosine kinetic isotope effects were determined using a gas chromatograph in conjunction with a mass selective 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'-(13)C]inosine and 1.113 +/- 0.008 for [1'-(2)H]inosine. The transition state consistent with these isotope effects is also consistent with the transition state proposed by Schramm and Horenstein using radioactive substrates.