Origin of Enzymatic Kinetic Isotope Effects in Human Purine Nucleoside Phosphorylase

ACS Catalysis ◽  
2017 ◽  
Vol 8 (2) ◽  
pp. 815-827 ◽  
Author(s):  
M. Roca ◽  
V. Moliner ◽  
I. Tuñón
Keyword(s):  
Purine Nucleoside Phosphorylase ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Purine Nucleoside ◽  
Nucleoside Phosphorylase ◽  
Kinetic Isotope

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.

scholarly journals Catalytic-site design for inverse heavy-enzyme isotope effects in human purine nucleoside phosphorylase

2017 ◽  
Vol 114 (25) ◽  
pp. 6456-6461 ◽  
Author(s):  
Rajesh K. Harijan ◽  
Ioanna Zoi ◽  
Dimitri Antoniou ◽  
Steven D. Schwartz ◽  
Vern L. Schramm
Keyword(s):  
Steady State ◽  
Transition State ◽  
Isotope Effect ◽  
Purine Nucleoside Phosphorylase ◽  
Isotope Effects ◽  
Catalytic Site ◽  
Purine Nucleoside ◽  
Barrier Crossing ◽  
Nucleoside Phosphorylase ◽  
Inverse Effect

Heavy-enzyme isotope effects (15N-, 13C-, and 2H-labeled protein) explore mass-dependent vibrational modes linked to catalysis. Transition path-sampling (TPS) calculations have predicted femtosecond dynamic coupling at the catalytic site of human purine nucleoside phosphorylase (PNP). Coupling is observed in heavy PNPs, where slowed barrier crossing caused a normal heavy-enzyme isotope effect (kchemlight/kchemheavy > 1.0). We used TPS to design mutant F159Y PNP, predicted to improve barrier crossing for heavy F159Y PNP, an attempt to generate a rare inverse heavy-enzyme isotope effect (kchemlight/kchemheavy < 1.0). Steady-state kinetic comparison of light and heavy native PNPs to light and heavy F159Y PNPs revealed similar kinetic properties. Pre–steady-state chemistry was slowed 32-fold in F159Y PNP. Pre–steady-state chemistry compared heavy and light native and F159Y PNPs and found a normal heavy-enzyme isotope effect of 1.31 for native PNP and an inverse effect of 0.75 for F159Y PNP. Increased isotopic mass in F159Y PNP causes more efficient transition state formation. Independent validation of the inverse isotope effect for heavy F159Y PNP came from commitment to catalysis experiments. Most heavy enzymes demonstrate normal heavy-enzyme isotope effects, and F159Y PNP is a rare example of an inverse effect. Crystal structures and TPS dynamics of native and F159Y PNPs explore the catalytic-site geometry associated with these catalytic changes. Experimental validation of TPS predictions for barrier crossing establishes the connection of rapid protein dynamics and vibrational coupling to enzymatic transition state passage.

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