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 reac tion is necessary for a complete description of XDH turnover with its presumed natural electron acceptor and to address the preference of WH for NAD over oxygen as a substrate. The reaction between pre-reduced XDH and NAD was studied by stopped-flow spectrophotometry. The reactio n was found to involve two rounds of oxidation with 2 eq of NAD. The f irst round goes to completion, and the second round reaches a slightly disfavored equilibrium. Rapid binding of NAD with an apparent K-d of 25 +/- 2 mu M 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) c ompletes a round of oxidation. These steps have been successfully test ed 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 equ ivalents. Rate constants for NAD reduction and NADH dissociation are r espectively 21- and 7-fold greater than k(cat), indicating that the re ductive half-reaction of the enzyme by xanthine is mostly rate-limitin g in xanthine/NAD turnover. A steady-state mechanism for XDH is discus sed.