Isotope, pulse-chase, stopped-flow, and rapid quench studies on the kinetic mechanism of bovine dihydropteridine reductase.
The kinetics of the reduction of quinonoid 2-amino-4-hydroxy-6, 7-dimethyldihydropteridine (DMPH2) catalyzed by bovine liver dihydropteridine reductase were examined with NADH, (S)-NADD, (S)-NADT, and [3H]-NADH as substrates. No significant deuterium isotope effect was observed on either Km or Vm, indicating that hydrogen transfer is not a major rate-limiting step of the reaction. Tritium from (S)-NADT is transferred to an exchangeable position of the pteridine product without significant isotopic discrimination. The ratio of tritium released into solvent to NAD+ produced is approximately 1.0 in the steady state as well as in the first enzyme turnover as determined by pulse-chase experiments. Pulse-chase methods also showed that the binary complex E.NADH is fully functional and can be completely converted to products prior to NADH dissociation in the presence of saturating DMPH2. The concentration of DMPH2 giving half-maximal trapping of E.NADH is identical with its Km as determined by steady-state kinetics. Stopped-flow kinetic measurements gave no evidence for a burst of NADH utilization. This was further demonstrated by rapid quench experiments which demonstrated a pre-steady-state rate nearly identical with that of the steady state. The above results are consistent with nonequilibrium ordered binding of substrates and with a rate-limiting isomerization in the ternary complex which precedes hydrogen transfer.
D000818:Animals; D002417:Cattle; D004093:Dihydropteridine Reductase / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D011621:Pteridines / Q000378:metabolism; D011865:Radioisotope Dilution Technique
D011621:Pteridines; D009243:NAD; C042293:quinonoid-2-amino-4-hydroxy-6,7-dimethyldihydropteridine; D004093:Dihydropteridine Reductase; D009247:NADH, NADPH Oxidoreductases