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Multi-site phosphorylation of pyruvate dehydrogenase complex

Multi-site phosphorylation of pyruvate dehydrogenase complex

FASEB Journal 15(4): A196, March 7

Inactivation of mammalian pyruvate dehydrogenase complex (PDC) depends upon phosphorylation of three serine residues (phosphorylation sites 1, 2, and 3) catalyzed by pyruvate dehydrogenase kinase (PDK). In animals there are at least four isozymes of kinase (PDK1, PDK2, PDK3, and PDK4). This study was undertaken in order to further explore the substrate specificity of isozymes. It was found that isozymes of kinase have markedly different specificities toward the individual phosphorylation sites of PDC. Only isozyme PDK1 was capable of phosphorylating all three phosphorylation sites. Under all conditions tested, the isozymes PDK2, PDK3, and PDK4 phosphorylated only sites 1 and 2. Furthermore, the rate of site 2 phosphorylation by isozyme PDK2 was by far lower than that for isozymes PDK3 and PDK4. If the holo-enzyme (TPP-bound PDC) was used as a kinase substrate, there was a dramatic decrease in the rates of phosphorylation of site 2 by PDK1 and PDK2, and of site 3 by PDK1. In contrast, TPP had lesser effect on the rates of phosphorylation of site 2 by PDK3 and PDK4. In fact, TPP inhibition of site 2 phosphorylation by PDK3 was observed only when the rates of phosphorylation reaction were determined at ATP concentration close to the Km value. The differences in the ability of isozymes of PDK to phosphorylate multiple phosphorylation sites had a great impact on the rates of re-activation of phospho-PDC by pyruvate dehydrogenase phosphatase (PDP). When PDC was inactivated as a result of PDK2 catalyzed phosphorylation, this phospho-PDC was readily re-activated by PDP. In contrast, multi-site phosphorylation of PDC carried out by isozymes PDK1, PDK3, and PDK4 markedly delayed the reactivation of the complex. Thus, results reported here strongly suggest that isozymes of PDK have different specificity toward the individual phosphorylation sites of PDC. This, in turn, greatly affects the rate of reactivation of phospho-PDC by PDP.

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Accession: 035349533

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