dsstructive 8guU m ischranir reperfusion m@y, and m normal physiological roles such as signal transduction. XOR is also able to act as an NADH oxidase m ...
Biochemical Society Transactions (1997) 25 517s 148 The reaction of humrn unthine dehydrogen8se With
NADE STEPHEN A. SANDERS, ROGER HARRISON, AND ROBERT EISENTHAL School of Biology and Biochemky, Bath University,Claverton Down, BATH,BA2 7AY, UK. XOR catalyses the oxidation of a wide range of substrates, most notably h y p o x d h e to xrathine, and xanthhe to uric acid. It exists m two intmeltible formq XDH, which prefersntiany uses NAD as reducing substnte, and XO, which uses molecular oxygen only. Reduction of molecular oxygen gives rise to the reauive 0species, superoxide and hydrogea peroxide. It is this property that has gmerated interest m human XOR as a potdsstructive 8guU m ischranir reperfusion m@y, and m normal physiological roles such as signal transduction. XOR is also able to act as an NADH oxidase m both its oxidase and dehydmgemse fonap [l-31. lhis ability, m Conjrmctioa with the low activity of human XOR &om some tissues towards h y p o x d h e and xnnthiue, has suggested a mechrrmgn * forXOR mediated i s c h a ~reperihdon injury which involves its NADH oxidase aotivity. A detailed investigation of the steady-state kinetics of NADH oxidase rctiVity of human and bovine XDH [3] has led to a proposed reaction tmchamm . involving a two-stage recycling of the enzyme. This mechanism was able to account for the observed substrate inhibition of superoxide production at high concentdons of NADH, and apparent MichaelisMenten kinetics for the rate ofNADH oxidation. In order to validate the proposed meohmism and to determine d i r d y some of the rate constants hived,the transient kinaics of the halfreaction of huuum XDH with NADH have been studied. XDH was purifies h m human milk as previously described [3]. The final product had a ratio of Azm to A450 of 5.2 and was >90% m the dehydrogenase form The enzyme solution was gel filtered into 50 mM Na+-phosphate b u l k pH 7.4 m an anaerobic glove box which was maintained at 1 ppm 02. Tke concentration of enzyme solutions was estimated using an extinction c o & h t at 450 nm of 37 mbf'm?.Solutions of NADH were made up m the same bulk on the day and standardised by their A340 u h g an * of 6.22 mW1m-'Kiuetic . studies were extinction cmfhent &ed out at 25.5 f 0.1OC ushg a rapid-scm stopped-flow apparatus with a diode may attachment m an anaerobic glove box maintained at 1 ppm 4. llbwrh-
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Figure 1. Absorbance spectra showing the reduction of human miUc XDH by NADH. Concentrations of enzyme and NADH were 4.9 pR4 and 15 pR4 respectively m 50 mM Na+-phosphate Mer pH 7.4. Spectra were rcoordcdaver 5.5 SecODds. Abbmhtions ueed: XOR, xnnthine oxidoteduotase; XDH, mthiue dehydmgemse; XO,xauthiue oxidase.
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Figure 2. The change m absorbance at 620 nm taken &omfigure 1. The solid line represents a k to abiphasicreactionwith rate constants of 33 s-' and 7 i'. The reaction of NADH with human XDH under anaerobic conditions was observed to be biphasic. The initial phase of the reaction involvres a decrease m absorbance at 450 nm, with a smaller increase m absorbance betwaa 550 and 650 nm (Figs 1 and 2), which is typical of reduction of fuy. oxidised flavin to a blue neutral flavin -one. The rate of this reaction is mdependent of NADH concentration m the rauge used (15 150 pM), with a first order rate constant of 33 s-'. As previously discussed [4], the greater increase m absorbance at 620 nm compared to 550 nm (Fig. 1) indicates at least partial reduction of another chromophore which absorbs at 550 nm, namely tin FdS centre. The seumd phase of the reaction results m a smaller decrease m absorbance at 450 nm, accompanied by a decrease in absorbance at 550 and 620 nm (Fii. 2). The decrease m &so during this phase of the reaction is approximatelytwice that of the decrease of 4 5 0 . These data are consistent with a Mer reduction of the Fe/S centres of the enzyme, accompanied by at least a partial reoxidation of the blue neutral flavin semiquinone. The rate constant for this phase of the reaction decreases fkom 7 3' at 15 phl NADH to 2.5 s" at 150 @ NADH. I An inverse relationship between NADH concentration and this rate constant for bovine XDH was attributed [4] to a stabilisation of the blue flavin semiquinoneby NADH The present data resemble those previously reported for bovine milk XDH by H u t and Massey [4]. The initinl rapid phase observed by Hunt and Massey m a triphasic reaction involves very small absorbance changes and may be beyond the resolution of the present data. This phase was attributed to the formation of a charge transfer complex between NADH and oxidised flavin. Qualitatively, the present data are similar to the last two phases of the triphasic reaction of NADH with bovine milk XDH, with a biphasic reduction m &SO and the appearance and disappearance of flavin semiquinone as indicated by the changes m &m. The overall decrease m absorbance at 450 nm however, is only 15% for human XDH (6.55% for bovine XDH), indicating that the h l level of reduction observed for human XDH is less than that observed for the bovine enzyme.
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1. Abadeh,S., Killacky,J., Bmboubetra,M., and Harrkm,R (1992) Biochim. Biophys. A&. 1117,25-32. 2. Sanders,S.A., Hanison,R, and Emthal,R (1996) Biochem. Soc.Trans.24,13S. 3. Sanders,S.A., Eisentbr/R, and H;lrrisolZR (1997) Eur. J. Biochem. submitted for publication 4. HuntJ. and Massey,V. (1994) J. BioL Chem. 269, 1890418914.
