The oxidative half-reaction of xanthine dehydrogenase with NAD; reaction kinetics and steady-state mechanism.

The reaction between reduced xanthine dehydrogenase (XDH) from bovine milk and NAD has been studied in detail. An understanding of this reaction is necessary for a complete description of XDH turnover with its presumed natural electron acceptor and to address the preference of XDH for NAD over oxygen as a substrate. The reaction between pre-reduced XDH and NAD was studied by stopped-flow spectrophotometry. The reaction was found to involve two rounds of oxidation with 2 eq of NAD. The first round goes to completion, and the second round reaches a slightly disfavored equilibrium. Rapid binding of NAD with an apparent Kd of 25 +/- 2 microM is followed by NAD reduction at a rate constant of 130 +/- 13 s-1. NADH dissociation at a rate constant of 42 +/- 12 s-1 completes a round of oxidation. These steps have been successfully tested and modeled to repeat themselves in the second round of oxidation. The association rate constant for NAD binding was estimated to be much greater than any rate constant measured in the oxidation by molecular oxygen, thus explaining how NAD competes with oxygen for reducing equivalents. Rate constants for NAD reduction and NADH dissociation are respectively 21- and 7-fold greater than kcat, indicating that the reductive half-reaction of the enzyme by xanthine is mostly rate-limiting in xanthine/NAD turnover. A steady-state mechanism for XDH is discussed.