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.
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