Transition state analysis of adenosine nucleosidase from yellow lupin ( Lupinus luteus)

Abstract

The transition state of adenosine nucleosidase (EC 3.2.2.7) isolated from yellow lupin ( Lupinus luteus) was determined based upon a series of heavy atom kinetic isotope effects. Adenosine labeled with 13C, 2H, and 15N was analyzed by liquid chromatography/electrospray mass spectrometry to determine kinetic isotope effects. Values of 1.024 ± 0.004, 1.121 ± 0.005, 1.093 ± 0.004, 0.993 ± 0.006, and 1.028 ± 0.005 were found for [1′- 13C], [1′- 2H], [2′- 2H], [5′- 2H], and [9- 15N] adenosine, respectively. Using a bond order bond energy vibrational analysis, a transition state consisting of a significantly broken C–N bond, formation of an oxocarbenium ion in the ribose ring, a conformation of C3- exo for the ribose ring, and protonation of the heterocyclic base was proposed. This transition state was found to be very similar to the transition state for nucleoside hydrolase, another purine metabolizing enzyme, isolated from Crithidia fasciculata.