Kinetic and chemical mechanisms of the sheep liver 6-phosphogluconate dehydrogenase
NE Price, PF Cook - Archives of biochemistry and biophysics, 1996 - Elsevier
NE Price, PF Cook
Archives of biochemistry and biophysics, 1996•ElsevierA complete kinetic characterization of sheep liver 6-phosphogluconate dehydrogenase
including product and dead-end inhibition patterns, primary deuterium isotope effects, and
the pH dependence of kinetic parameters has been completed in order to determine the
kinetic mechanism and obtain information on the chemical mechanism of the enzyme. A
rapid equilibrium random kinetic mechanism has been proposed, with product and dead-
end inhibition patterns both being symmetric. Ribulose 5-phosphate and 6-sulfogluconate …
including product and dead-end inhibition patterns, primary deuterium isotope effects, and
the pH dependence of kinetic parameters has been completed in order to determine the
kinetic mechanism and obtain information on the chemical mechanism of the enzyme. A
rapid equilibrium random kinetic mechanism has been proposed, with product and dead-
end inhibition patterns both being symmetric. Ribulose 5-phosphate and 6-sulfogluconate …
A complete kinetic characterization of sheep liver 6-phosphogluconate dehydrogenase including product and dead-end inhibition patterns, primary deuterium isotope effects, and the pH dependence of kinetic parameters has been completed in order to determine the kinetic mechanism and obtain information on the chemical mechanism of the enzyme. A rapid equilibrium random kinetic mechanism has been proposed, with product and dead-end inhibition patterns both being symmetric. Ribulose 5-phosphate and 6-sulfogluconate are both competitive with 6-phosphogluconate (6-PG) and noncompetitive with NADP, and NADPH and ATP-ribose are both competitive with NADP and noncompetitive with 6-phosphogluconate. Equal primary deuterium isotope effects of 1.5–2 onDV,DV/KNADP, andDV/K6-PGwith 3-deuterio-6-PG confirm a rapid equilibrium random mechanism and show that hydride transfer is at least partially rate limiting in the overall reaction. The maximum velocity is pH dependent, decreasing at low and high pH with slopes of 1 and −1, respectively, and pKvalues of 6.4 and 8.6. TheV/KNADPandV/K6-PGalso decrease at low and high pH with slopes of 1 and −1, giving pKvalues of 6.8 and 8.7 and of 6.9 and 7.8, respectively. The pH rate profiles are consistent with a general acid/general base mechanism where the catalytic residues are involved in binding. Reverse protonation states between the general acid and the general base are proposed where an unprotonated general base accepts a proton from the C-3 hydroxyl of 6-PG concomitant with hydride transfer followed by decarboxylation of the resulting 3-keto intermediate to give an enediol which is protonated by the general acid to form ribulose 5-phosphate. The pH dependence of the pKiprofile of the inhibitory analog 5-phosphoribonate decreases at low and high pH with slopes of 1 and −1, respectively, and pKs of 6.2 and 7.4 and suggests that intrinsic pKs are observed in theV/Kprofiles. The pKs of both the general base and general acid in the E:6-PG complex appears to be perturbed such that the general base decreases from 7.4–7.8 to a value of 6.4–6.8, and the pKof the general acid increases from 6.2–6.9 to a value of 8.6–8.7, as a result of direct interaction with 6PG. Data are interpreted with regard to the published crystal structures of the E:6-PG, E:NADP, and E:NADPH complexes.
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