Interaction of thiamin diphosphate with phosphorylated and dephosphorylated mammalian pyruvate dehydrogenase complex
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Xiaoqing Liu
Abstract
Kinetic and binding studies were carried out on substrate and cofactor interaction with the pyruvate dehydrogenase complex from bovine heart. Fluoropyruvate and pyruvamide, previously described as irreversible and allosteric inhibitors, respectively, are strong competitive inhibitors with respect to pyruvate. Binding of thiamin diphosphate was used to study differences between the active dephosphorylated and inactive phosphorylated enzyme states by spectroscopic methods. The change in both the intrinsic tryptophan fluorescence and the fluorescence of the 6-bromoacetyl-2-dimethylaminonaphthalene-labelled enzyme complex produced on addition of the cofactor showed similar binding behaviour for both enzyme forms, with slightly higher affinity for the phosphorylated form. Changes in the CD spectrum, especially the negative Cotton effect at 330 nm as a function of cofactor concentration, both in the absence and presence of pyruvate, also revealed no drastic differences between the two enzyme forms. Thus, inactivation of the enzyme activity of the pyruvate dehydrogenase complex is not caused by impeding the binding of substrate or cofactor.
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Articles in the same Issue
- The structure of the juvenile hormone binding protein gene from Galleria mellonella
- Interaction of thiamin diphosphate with phosphorylated and dephosphorylated mammalian pyruvate dehydrogenase complex
- Purification and characterization of a chitinase-like antifungal protein from black turtle bean with stimulatory effect on nitric oxide production by macrophages
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- GRIP1 mediates the interaction between the amino- and carboxyl-termini of the androgen receptor
- Dual concentration-dependent activity of thyroglobulin type-1 domain of testican: specific inhibitor and substrate of cathepsin L
- Proteolysis of insulin-like growth factor binding proteins (IGFBPs) by calpain