[Role of muscle pyruvate dehydrogenase histidine residues in thiamine pyrophosphate binding].
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.
D002384:Catalysis; D004047:Diethyl Pyrocarbonate; D004228:Dithionitrobenzoic Acid; D004229:Dithiothreitol; D004793:Enzyme Reactivators; D006639:Histidine; D006898:Hydroxylamines; D008274:Magnesium; D011768:Pyruvate Dehydrogenase Complex / Q000037:antagonists & inhibitors; D013835:Thiamine Pyrophosphate
D004793:Enzyme Reactivators; D006898:Hydroxylamines; D011768:Pyruvate Dehydrogenase Complex; D006639:Histidine; D004228:Dithionitrobenzoic Acid; D008274:Magnesium; D004047:Diethyl Pyrocarbonate; D013835:Thiamine Pyrophosphate; D004229:Dithiothreitol
Rol' ostatkov gistidina myshechnoĭ piruvatdegidrogenazy v sviazyvanii tiaminpirofosfata