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Xanthine oxidase (XO) and xanthine dehydrogenase (XDH) are widespread enzymes that perform a critical step in the catabolism of purine nucleotides. These enzymes oxidize xanthine, the catabolic product of purine nucleotide degradation, to uric acid, which is subsequently excreted or further degraded depending on the organism. The oxidation of xanthine is performed by cleaving the C-H bond at the 8 position of xanthine, and two electrons are donated to a Mo (VI) ion in the enzyme active site while the corresponding proton is released to an unidentified enzyme ligand of the molybdenum ion (1). The electrons rapidly equilibrate in the enzyme between the Mo (IV) ion, two Fe-S centers, and a flavin. After cleavage of the C-H bond, the substrate is hydroxylated to an enol by water in the solvent, and both the product and the proton released from the 8-position pass from the active site into the solvent. The only significant difference between the mechanism of XO and XDH is the oxidative process at the flavin cofactor by which the enzyme is restored to its original state; the enzyme is oxidized in xanthine oxidase by reducing molecular oxygen to hydrogen peroxide, whereas in XDH the oxidant is FAD. Numerous kinetic and mechanistic studies have been performed on these enzymes, particularly on bovine milk XO and chicken liver XDH, and the rate limiting step for the entire catalytic process has been shown to be the release of the product from the enzyme. (1) At present, however, few details are known about the mechanism by which this process occurs. This study utilized kinetic assays of bovine milk XO, chicken liver XDH, and C. acidovorans XDH in the presence of normal and deuterated aqueous solvents and with regular- and [8]deutero-xanthine substrate. It was hoped that the effect of deuterated solvent on the steady state reaction rate would elucidate any role that solvent-derived protons play in the rate limiting step of product release. Furthermore, comparisons between XO and XDH were facilitated by using enzymes from several organisms, including the prokaryotic bacterium C. acidovorans.
Kinetic assays were performed on a Beckman Model 2400 spectrophotometer with a 1 cm light path at 25š C in 50 mM potassium phosphate, 1 mM EDTA, pH 7.7. Absorbance was monitored at 295 nm using an extinction coefficient for uric acid of 9.6 mM-1 cm-1. Xanthine solutions were prepared from a stock solution containing 0.03 M xanthine and 0.04 M NaOH, and 0.5 mM NAD was included in the XDH assay solutions. [8-D]xanthine was provided by the Edmondson laboratory and prepared in D2O with NaOD, and the deuterium oxide solvent was prepared by rotary evaporating the aqueous solvent and replacing it with 99.9% D2O. All chemical reagents were obtained from Sigma Chemical company, and all three enzymes were previously purified in the Edmondson laboratory. Kinetic data was calculated by Enzfitter using non-linear regression analysis for Michaelis-Menten kinetics.
The solvent isotope effects and [8-D]xanthine isotope effects on all of the enzymes are quite small, and this makes it very difficult to distinguish errors from legitimate isotope effects. There are no striking trends in the solvent isotope effects on all three enzymes. The ratios between the maximum rate of the enzymes in water and in D2O are as follows in regular substrate: XO, 2.11; C. acid. XDH, 1.17; chick. XDH, 0.91; in deuterated xanthine: XO, 1.66; C. acid. XDH, 1.15, chick. XDH, 1.42. It does appear that the steady state maximum rate for xanthine oxidase is consistently affected to a much greater extent than the rate of the dehydrogenases when D2O is used as solvent. The Km and V/K solvent isotope effects, however, do not follow the same pattern and differ among the enzymes.
The Km values obtained with [8-D] xanthine may be in error because of salt impurities in the compound which led to errors in weighing accurate solution concentrations. This is especially noticeable for the ratio between [8-H]- and [8-D]xanthine kinetics (H/D) in water on C. acidovorans XDH, for which there are very different results for the original [8- D]xanthine solution and the second solution that was prepared. The (H/D) data for the second, more reliable substrate solution do reveal a significant 2- to 3-fold effect on the Vmax and perhaps a 2-fold effect on the Kcat and V/K values for the C. acidovorans dehydrogenase.
The most reliable kinetic isotope data obtained in this study appears to be the solvent isotope effect on each enzyme with [8-H]xanthine substrate.
It appears that solvent-derived protons are involved in the product release step in xanthine oxidase, as evidenced by the halving of the overall steady state reaction rate in D2O. It also appears that this may not be the case for the dehydrogenases, for which D2O solvent had little effect.
- D'Ardenne, S. C., & Edmondson, D. E. (1990) Biochemistry 29, 9046.
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