scholarly journals Investigation of α-deuterium kinetic isotope effects on the purine nucleoside phosphorylase reaction by the equilibrium-perturbation technique

1989 ◽  
Vol 257 (2) ◽  
pp. 355-359 ◽  
Author(s):  
P K Lehikoinen ◽  
M L Sinnott ◽  
T A Krenitsky
Keyword(s):  
Escherichia Coli ◽  
Equilibrium Constant ◽  
Purine Nucleoside Phosphorylase ◽  
Perturbation Technique ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Purine Nucleoside ◽  
Nucleoside Phosphorylase ◽  
Kinetic Isotope

1. alpha-Deuterium kinetic isotope effects on the phosphorolysis of inosine catalysed by Escherichia coli purine nucleoside phosphorylase were measured by the equilibrium-perturbation technique, by using the change in absorbance at 250 nm (approx. 20%). 2. Values of 2H(V/K) of 1.13(9) at pH 5.0, 1.10(5) at pH 6.1, 1.09(4) at pH 7.3, 1.08 at pH 8.4 and 1.16(4) at pH 9.4 were obtained. 3. These are compared with literature alpha-deuterium kinetic isotope effects for this and related reactions. 4. The equilibrium constant, defined as [inosine].[H2PO4-]/[hypoxanthine] [alpha-Rib f OPO3H-], is 46 at 25 degrees C. 5. N-3-beta-D-Ribofuranosylhypoxanthine, an impurity in chemically synthesized inosine, is a substrate.

scholarly journals β-deuterium kinetic isotope effects in the purine nucleoside phosphorylase reaction

1991 ◽  
Vol 278 (2) ◽  
pp. 487-491 ◽  
Author(s):  
X M Guo ◽  
M Ashwell ◽  
M L Sinnott ◽  
T A Krenitsky
Keyword(s):  
Transition State ◽  
Purine Nucleoside Phosphorylase ◽  
Perturbation Technique ◽  
Isotope Effects ◽  
Purine Nucleoside ◽  
Ph Optimum ◽  
Nucleoside Phosphorylase ◽  
Ph Variation ◽  
Kinetic Isotope ◽  
And Inversion

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.

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