Kozachenko A I, Nagler L G, Vartanian L S
Biokhimiia. 1976 Feb;41(2):349-56.
Kinetic characteristics for reactivity of SH-groups of milk xanthine oxidase were obtained under different conditions. Two types of SH-groups with rate constant values, differing by a factor of about 50, were found in a phosphate buffer at pH 7.0. The slow stage of reaction is followed by protein precipitation. The number of fast- (12) and slowly-reacting (60) groups were calculated from the kinetic data. The blocking of the fast-reacting groups occurs without loss of the enzyme activity. The values of activation energy for the fast- and slowly-reacting groups are 15 and 48 kcal/mol respectively. The formation of the enzyme-substrate complex stabilizes the enzyme molecule; the number of fast-reacting SH-groups and the rate constant values for both types of groups remain unchanged, whereas the number of slowly-reacting SH-groups markedly decreases (37). The values of activation energy for both types of SH-groups show no changes in the presence of substrate. Conformations of the enzyme in different denaturating solvents were characterized by a number of SH-groups, reacting with p-chloromercurybenzoate. 54 groups are exposed in solutions of groups exposed in 7.0-8.5 M urea solutions is 35-38. In all solvents studied the protein molecule is probably not completely unfolded, since the number of exposed SH-groups is less than the full number of SH-groups determined by the amino acid analysis. Only 42 SH-groups reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) under the same conditions.
在不同条件下获得了牛奶黄嘌呤氧化酶SH基团反应性的动力学特征。在pH 7.0的磷酸盐缓冲液中发现了两种类型的SH基团，其速率常数相差约50倍。反应的缓慢阶段之后是蛋白质沉淀。根据动力学数据计算出快速反应（12个）和缓慢反应（60个）基团的数量。快速反应基团的封闭在不损失酶活性的情况下发生。快速反应基团和缓慢反应基团的活化能值分别为15和48千卡/摩尔。酶-底物复合物的形成使酶分子稳定；快速反应SH基团的数量以及两种类型基团的速率常数不变，而缓慢反应SH基团的数量显著减少（37个）。在有底物存在的情况下，两种类型SH基团的活化能值没有变化。通过与对氯汞苯甲酸反应的SH基团数量来表征酶在不同变性溶剂中的构象。在7.0 - 8.5 M尿素溶液中暴露的基团数量为35 - 38个。在所有研究的溶剂中，蛋白质分子可能没有完全展开，因为暴露的SH基团数量少于通过氨基酸分析确定的SH基团总数。在相同条件下，只有42个SH基团与5,5'-二硫代双（2-硝基苯甲酸）反应。
