beta-deuterium kinetic isotope effects in the purine nucleoside phosphorylase reaction

Biochem J. 1991 Sep 1;278 ( Pt 2)(Pt 2):487-91. doi: 10.1042/bj2780487.


1. [2'-2H]Inosine was made from inosine by tetraisopropyldisiloxanyl protection of the 3'- and 5'-positions, oxidation with dimethyl sulphoxide and acetic anhydride, immediate NaB2H4 reduction of the oxo sugar product and inversion at C-2' of the resultant protected [2'-2H]arabino-inosine by trifluoromethanesulphonylation and reaction with caesium propionate, followed by deprotection. 2. The equilibrium-perturbation technique was used to measure beta 2H(V/K) for phosphorolysis of this compound by the purine nucleoside phosphorylase of Escherichia coli as a function of pH. 3. The pH variation indicates an intrinsic effect of 1.068 masked by isotopically silent steps near the pH optimum. 4. The similar pH variation of these beta-deuterium effects and the alpha-deuterium effects measured previously [Stein & Cordes (1981) J. Biol. Chem. 256, 767-772; Lehikoinen, Sinnott & Krenitsky (1989) Biochem. J. 257, 355-359] for this reaction provides the first experimental reassurance for the common assumption that pH changes merely mask and unmask the chemical steps in an enzyme-catalysed reaction, and do not detectably alter transition-state structure. 5. The dihedral angle between the C-H-2' bond and the electron-deficient p-orbital at the transition state is in the range 32-48 degrees, in accord with an essentially planar furanose ring.

Publication types

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

MeSH terms

  • Deuterium / chemistry*
  • Escherichia coli / enzymology
  • Hydrogen-Ion Concentration
  • Inosine / chemistry
  • Isotopes
  • Kinetics
  • Purine-Nucleoside Phosphorylase / chemistry*
  • Spectrophotometry, Ultraviolet


  • Isotopes
  • Inosine
  • Deuterium
  • Purine-Nucleoside Phosphorylase