Transient and steady-state kinetic analyses of xanthine oxidoreductase.

Xanthine oxidase (XO) and xanthine dehydrogenase (XDH) from bovine milk are interconvertible forms of the same enzyme. The proteins differ in respect to the number of cysteines versus cystines. Cysteine oxidation on conversion of XDH to XO results in a conformational change with profound effects on the enzyme, most notably a change in the preferred oxidizing substrate. XDH prefers NAD, but will use oxygen in the absence of NAD; XO only reacts significantly with oxygen. These studies help indicate how changes in protein structure can modulate the chemistry of an FAD cofactor. Also, characterizing the reactivity of XDH towards oxygen may aid in clarifying the role of XO and XDH in several oxidative pathologies. Studies of the reductive half-reaction of XO and XDH with the alternate substrate 4-hydroxypyrimidine indicate there is little change at the molybdenum site. Two different intramolecular electron transfer reactions were resolved. Kinetic isotope effects suggest the first electron transfer is under prototropic control. The second electron transfer appears to be triggered by dissociation of the bound product. Reactivity of reduced XDH toward oxygen was shown to be inherent to XDH and not due to contaminating XO. In the absence of NAD, xanthine/oxygen and NADH/oxygen turnover occur at 33% and 40% the k$\\rm \\sb{cat}$ of xanthine/NAD turnover, respectively. To determine the basis of substrate specificity, quantitative mechanisms were elucidated of how XDH reacts with NAD and with molecular oxygen. The estimated second-order rate constant for NAD binding is 560-fold greater than the largest rate constant of the oxygen reaction. Consistent with these predictions, oxygen was measured to be very poor at inhibiting the initial rate of xanthine/NAD turnover; however, substantial oxygen consumption was observed at longer reaction times. Conditions are described under which XDH-catalyzed formation of reactive oxygen species is likely. Determinants of substrate specificity between XO and XDH were also studied. The role of the lower FAD midpoint potential in XDH was investigated by substitution with artificial flavins. Spectral perturbations on addition of NAD and the strength of NAD binding are compared between XO and XDH. The NAD binding site in XO is still intact, but it places NAD in a non-productive orientation, away from the FAD.