Nishino T, Tsushima K, Hille R, Massey V
J Biol Chem. 1982 Jul 10;257(13):7348-53.
Milk xanthine oxidase reacted with fluorodinitrobenzene resulting in the modification of two lysine residues with a 6-fold decrease in catalytic activity. Continued reaction with fluorodinitrobenzene up to a total of 11 dinitrophenyl residues/equivalent of enzyme-bound FAD resulted in no further decrease in activity. Stopped flow studies revealed that the modification perturbed the reduction of the enzyme by xanthine; this was 6-fold lower with modified than with native enzyme. The reaction of the reduced modified enzyme with oxygen was qualitatively and quantitatively the same as with native enzyme. One nitro group of each dinitrophenyl lysine residue is slowly reduced by xanthine; reduction of both nitro groups is achieved by dithionite. The two dinitrophenyl lysine reduces can be distinguished on the basis of their kinetics of reduction. One appears to be located on the protein surface and is reduced in an intermolecular reaction, while the other appears to be located in a pocket of the enzyme and is reduced in a slow intramolecular reaction.
牛奶黄嘌呤氧化酶与氟二硝基苯反应，导致两个赖氨酸残基被修饰，催化活性降低了6倍。继续与氟二硝基苯反应，直至每当量酶结合的FAD共有11个二硝基苯基残基，活性不再进一步降低。停流研究表明，这种修饰干扰了黄嘌呤对酶的还原作用；修饰后的酶的还原作用比天然酶低6倍。还原型修饰酶与氧气的反应在定性和定量上与天然酶相同。每个二硝基苯基赖氨酸残基的一个硝基被黄嘌呤缓慢还原；连二亚硫酸盐可实现两个硝基的还原。两个二硝基苯基赖氨酸的还原可以根据它们的还原动力学来区分。一个似乎位于蛋白质表面，在分子间反应中被还原，而另一个似乎位于酶的一个口袋中，在缓慢的分子内反应中被还原。
