Acid base catalytic mechanism of the dihydropyrimidine dehydrogenase from pH studies.
Primary deuterium (NADPH(D)), solvent deuterium, and multiple isotope effects and the pH dependence of kinetic parameters have been used to probe the mechanism of the dihydropyrimidine dehydrogenase from pig liver. Isotope effect and pH-rate data suggest a rate-determining reductive half-reaction in which reduction of the flavin by NADPH has only a minor rate limitation (DV approximately D(V/KNADPH) approximately 1.1), while protonation of the flavin at N-1 occurring in a step following reduction is slow (D2OV = 3, while D2O(V/KNADPH) = 2). An enzymatic general acid with a pK of 8.2 is required to protonate N-1 of the flavin. In the second half-reaction, uracil is reduced at C-6 by flavin and protonated on the opposite face at C-5 by an enzymatic general acid with a pK of 9. The hydride transfer from N-5 of the flavin to C-5 of uracil is facilitated by an enzymatic general base with a pK of 5.6 that accepts a proton from N-1 of the flavin. There is also evidence from the pH dependence of V and the V/K for reduced dinucleotide substrates that a second enzyme residue with a pK of 6.4 must be unprotonated for optimum activity, but is not essential for activity. None of the functional groups reflected in the V/KNADPH pH-rate profile have a role in binding, while both of those observed in the V/Kuracil profile have a role in binding as shown by the pH dependence of the dissociation constants for the competitive inhibitors ATP-ribose and 2,6-dihydroxypyridine.
Podschun
B
B
Theodor-Boveri-Institut für Biowissenschaften (Biozentrum), Universität Würzburg, Germany.
Jahnke
K
K
Schnackerz
K D
KD
Cook
P F
PF
D000818:Animals; D003600:Cytosol / Q000201:enzymology; D003903:Deuterium; D042943:Dihydrouracil Dehydrogenase (NADP); D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D008961:Models, Structural; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D013552:Swine
D009249:NADP; D003903:Deuterium; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP)