The mechanism of regulatory phosphorylation of the pyruvate dehydrogenase component (El) of muscle pyruvate dehydrogenase complex was studied. Chemical modification of the arginine residues essential for substrate binding was shown to prevent incorporation of 32p from [T-3zP]ATP into E1 catalyzed by kinase and to exclude completely the interaction of holo-E1 with pyruvate. It is proposed that negatively charged phosphoseryl residues may compete with pyruvate for the active site arginine and thereby block the substrate binding.presence of [7-:~2p]ATP. Aliquots were removed and either assayed for PDC (El) activity or spotted on 2 × 2 cm Whatman 3MM paper and, following further treatment, counted for protein-bound radioactivity according to [11]. Protein was determined by the method of Bradford [12]. Arginine residues were modified with 2,3-butanedione, phenylglyoxal (PG) and 4-OH-3-NO2-PG as described earlier [13,14]. Unreacted diketone reagents were removed by the gel-filtration technique. Kinase action in these experiments was controlled after combining native or modified El with saturating concentrations of the E2-X-kinase fraction. Circular dichroism spectra were recorded on a CNRS-Russel-Jouan Dichrograph 1 ! 1 as described earlier [2].
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