Lane R S, Hansen B A, Dekker E E
Biochim Biophys Acta. 1977 Mar 15;481(1):212-21. doi: 10.1016/0005-2744(77)90153-x.
Bovine liver 2-oxo-4-hydroxyglutarate aldolase (suggested name: 2-oxo-4-hydroxyglutarate glyoxylate-lyase catalyzing the reaction: 2-oxo-4-hydroxyglutarate in equilibrium pyruvate + glyoxylate) contains eight to ten sulfhydryl groups as determined by titration of the enzyme with either 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) or p-mercuribenzoate in the presence of 1% sodium dodecyl sulfate. In the absence of a denaturant, all of the cysteinyl residues react with p-mercuribenzoate whereas only four are accessible to titration with Nbs2. No differences in -SH group reactivity can be detected during titration of the aldolase with p-mercuribenzoate. In contrast, two classes of sulfhydryls can be differentiated in the disulfide exchange reaction with Nbs2 in the absence of a denaturant; one -SH group (Class I) reacts rapidly whereas three additional thiols (Class II) titrate at approx. 0.1 the rate of the Class I-SH residue. Both pyruvate and glyoxylate protect one of the three -SH residues in Class II from reaction with Nbs2. Either substrate also prevents titration of one to two thiol groups by p-mercuribenzoate and decreases the rate of reaction of aldolase -SH groups with Nbs2 in 8 M urea. These ligand-induced changes in -SH reactivity provide a sensitive indication that the enzyme exists in an altered conformational state in the presence of either of its cosubstrates. Titration of the enzyme with either Nbs2 or p-mercuribenzoate results in a progressive loss of aldolase activity which is not proportional to the number of -SH groups modified. The enzyme retains 50% of the activity of the native enzyme when Class I and Class II thiols (i.e. four -SH groups total) are modified with Nbs2; 15% residual activity is still observed following titration of all of the cysteinyl residues with p-mercuribenzoate. Pyruvate and glyoxylate provide partial protection against inactivation. It is concluded that inactivation of 2-oxo-4-hydroxyglutarate aldolase by Nbs2 or p-mercuribenzoate is a consequence of alterations in protein structure which accompany modification of -SH groups. The data argue against the direct participation of an active-site thiol group in the catalytic mechanism of 2-oxo-4-hydroxyglutarate aldolase, be that aldol cleavage and condensation or beta-decarboxylation.
牛肝2-氧代-4-羟基戊二酸醛缩酶（建议名称：催化反应2-氧代-4-羟基戊二酸⇌丙酮酸+乙醛酸的2-氧代-4-羟基戊二酸乙醛酸裂解酶）含有8至10个巯基，这是通过在1%十二烷基硫酸钠存在下用5,5'-二硫代双（2-硝基苯甲酸）（Nbs2）或对氯汞苯甲酸滴定该酶来确定的。在没有变性剂的情况下，所有半胱氨酰残基都与对氯汞苯甲酸反应，而用Nbs2滴定只能检测到四个可及的巯基。在用对氯汞苯甲酸滴定醛缩酶的过程中，未检测到巯基反应性的差异。相反，在没有变性剂的情况下，在与Nbs2的二硫键交换反应中可以区分出两类巯基；一个巯基（I类）反应迅速，而另外三个硫醇（II类）的滴定速率约为I类巯基残基的0.1倍。丙酮酸和乙醛酸都能保护II类中的三个巯基之一不与Nbs2反应。任何一种底物还能阻止对氯汞苯甲酸滴定一到两个巯基，并降低醛缩酶巯基在8M尿素中与Nbs2的反应速率。这些配体诱导的巯基反应性变化提供了一个敏感的迹象，表明在其任何一种共底物存在下，该酶以改变的构象状态存在。用Nbs2或对氯汞苯甲酸滴定该酶会导致醛缩酶活性逐渐丧失，这与被修饰的巯基数量不成比例。当I类和II类硫醇（即总共四个巯基）用Nbs2修饰时，该酶保留了天然酶50%的活性；在用对氯汞苯甲酸滴定所有半胱氨酰残基后，仍观察到15%的残余活性。丙酮酸和乙醛酸提供部分抗失活保护。可以得出结论，Nbs2或对氯汞苯甲酸使2-氧代-4-羟基戊二酸醛缩酶失活是蛋白质结构改变的结果，这种改变伴随着巯基的修饰。数据表明，活性位点巯基不直接参与2-氧代-4-羟基戊二酸醛缩酶的催化机制，无论是醛醇裂解和缩合还是β-脱羧反应。
