In order to demonstrate the role of the protein component of pyruvate decarboxylase in the mechanism of substrate activation, we have isolated and characterized two states of the enzyme, the non-activated and the substrate-activated state, by covalent linking with bifunctional reagents. Because of the fact that modification of the reactive amino groups by 2,4,6-trinitobenzenesulfonic acid or methyl propionimidate influences neither the catalytic nor the regulatory properties of pyruvate decarboxylase, we used bisimidates of different chain length in the modification experiments. Both the non-activated and the substrate-activated enzyme states could be characterized separately. The lag phase of product formation as a typical property of the native enzyme disappeared completely when the enzyme had been cross-linked in the presence of the substrate. The permanently activated enzyme state shows 85% of the activity of native pyruvate decarboxylase and is exclusively stabilized by intra-subunit links. Elimination and subsequent reincorporation of the cofactors thiamine pyrophosphate and magnesium ions resulted in a complete regaining of the properties of the permanently activated enzyme form. An inactive enzyme form was obtained after cross-linking of non-activated pyruvate decarboxylase at low ionic strength (less than 0.01). Using a disulfide-containing linker we could prove that the inactivity of the obtained enzyme preparation was only the result of the incorporated cross-links and not that of denaturation.

• PMID: 2275720
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Journal Article

Authors

König S, Hübner G, Schellenberger A

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