Abstract
We report results of steady state kinetic studies of dihydropteridine reductase from bovine liver. Initial velocity analysis excluded the possibility of a rapid equilibrium ordered mechanism, but could not decisively differentiate between a ping-pong mechanism and a sequential mechanism. Product inhibition patterns with NAD + were competitive against NADH and noncompetitive against quinonoid dihydropterin, thus excluding a ping-pong mechanism and a rapid equilibrium random bi-bi mechanism. The possibility that a dead-end complex is formed between enzyme, pterin, and NAD + in this last mechanism was excluded by the observation that the dead-end inhibitor, aminopterin, is uncompetitive against NADH and noncompetitive against the pterin. Another dead-end inhibitor, Cibacron blue 3G-A, was found to be competitive against NADH and noncompetitive against the pterin. We conclude that the kinetic mechanism of dihydropteridine reductase is ordered bi-bi with the binding of NADH first and the release of NAD + last.
Published Version
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