Severin S E, Khaĭlova L S, Kereeva D N
Ukr Biokhim Zh. 1976 Jul-Aug;48(4):510-6.
The interaction of diethylpyrocarbonate (DEP) with the pyruvate dehydrogenase component (PDH) isolated from the pyruvate dehydrogenase complex (EC 1.2.4.1) results in a modification of 3-5 histidine residues per mole of enzyme, which simultaneously decreases the enzyme activity. After PDH inhibilion by DEP in the presence of dithiothreitol almost complete reactivation (94%) under the effect of neutral hydroxylamine is observed. In the absence of SH-groups protection incomplete reactivation by hydroxylamine (79%) is found. In the latter case titration with 5,5-dithio--bis-(2-nitrobenzoic acid) in 8 M urea showed that the DEP-modified protein contains less quantity of SH groups (by 4-8) as compared to the native enzyme. It is assumed that the DEP-modified SH-groups are not responsible for the enzyme activity. The differential spectrum of the modified and native PDH showed no changes within the range of 260-300 nm. TPP in combination with Mg2+ (10(-3) M) protectes PDH from being inactivated by DEP. TPP (10(-2) M) reactivates PDH by 70% after its complete inhibition by DEP. Similar protective action is manifested by ATP, ADP and inorganic pyrophosphate in the presence of Mg2+. A kinetic study showed a competitive type of PDH inhibition by DEP with respect to TPP. it is concluded that the histidine residues of PDH are involved in TPP binding.
焦碳酸二乙酯（DEP）与从丙酮酸脱氢酶复合体（EC 1.2.4.1）中分离出的丙酮酸脱氢酶组分（PDH）相互作用，导致每摩尔酶有3 - 5个组氨酸残基被修饰，同时酶活性降低。在二硫苏糖醇存在的情况下，DEP抑制PDH后，在中性羟胺的作用下几乎可完全重新激活（94%）。在没有SH基团保护的情况下，发现羟胺的重新激活不完全（79%）。在后一种情况下，在8 M尿素中用5,5 - 二硫代 - 双 -（2 - 硝基苯甲酸）滴定表明，与天然酶相比，DEP修饰的蛋白质含有的SH基团数量更少（少4 - 8个）。据推测，DEP修饰的SH基团与酶活性无关。修饰后的和天然的PDH的差分光谱在260 - 300 nm范围内没有变化。TPP与Mg2 +（10(-3) M）结合可保护PDH不被DEP灭活。在PDH被DEP完全抑制后，TPP（10(-2) M）可使PDH重新激活70%。在Mg2 +存在的情况下，ATP、ADP和无机焦磷酸也表现出类似的保护作用。动力学研究表明，DEP对PDH的抑制作用相对于TPP呈竞争性。得出的结论是，PDH的组氨酸残基参与TPP的结合。
