Phosphorylation and inactivation of the pyruvate dehydrogenase from the anaerobic parasitic nematode, Ascaris suum. Stoichiometry and amino acid sequence around the phosphorylation sites.
Tryptic digestion of the fully phosphorylated Ascaris suum pyruvate dehydrogenase complex yielded a single tetradecapeptide containing 2 phosphorylated serine residues. Its amino acid sequence was Tyr-Ser-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Ser(P)-Tyr-Arg and was very similar to one of the tryptic phosphopeptides isolated from mammalian and yeast pyruvate dehydrogenases. At partial phosphorylation, three peptides were isolated which corresponded to the monophosphorylated (sites 1 and 2) and diphosphorylated tetradecapeptides. In contrast to results reported from mammalian complexes, phosphorylation of the ascarid complex paralleled inactivation, and no additional phosphorylation occurred after inactivation was complete. Complete inactivation of the complex was associated with the incorporation of 1.7-1.9 mol of phosphoryl groups/mol of alpha-pyruvate dehydrogenase subunit, and the strict preference of the pyruvate dehydrogenase kinase for site 1 was not observed. Whereas site 1 was initially phosphorylated more rapidly than site 2, at 50% inactivation, 41% of the incorporated phosphoryl groups were incorporated into site 2. In addition, substantial amounts of peptide monophosphorylated at site 2 also accumulated, suggesting that prior phosphorylation at site 1 was not necessary for phosphorylation at site 2. Phosphorylation also caused a marked decrease in the mobility of the alpha-pyruvate dehydrogenase subunit on sodium dodecyl sulfate-polyacrylamide gels and the apparent separation of mono- and diphosphorylated forms of the enzyme. The significance of these observations in the regulation of the unique anaerobic mitochondrial metabolism of A. suum is discussed.
Thissen
J
J
Department of Biology, University of Toledo, Ohio 43606.
Komuniecki
R
R
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D000818:Animals; D001200:Ascaris / Q000201:enzymology*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D011768:Pyruvate Dehydrogenase Complex; D014357:Trypsin