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.
Research Article| September 01 1991
β-deuterium kinetic isotope effects in the purine nucleoside phosphorylase reaction
X M Guo;
M L Sinnott;
Biochem J (1991) 278 (2): 487-491.
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X M Guo, M Ashwell, M L Sinnott, T A Krenitsky; β-deuterium kinetic isotope effects in the purine nucleoside phosphorylase reaction. Biochem J 1 September 1991; 278 (2): 487–491. doi: https://doi.org/10.1042/bj2780487
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