Proteinase-catalyzed activation of porcine heart muscle pyruvate dehydrogenase and identification of its cleavage site.
Porcine heart muscle pyruvate dehydrogenase (PDH, EC 1.2.4.1) with subunit composition alpha 2 beta 2 catalyzes the initial decarboxylation step of an oxidative decarboxylation sequence of pyruvate. Highly purified PDH, was further activated several-fold by limited digestion with trypsin, Staphylococcus aureus V8 proteinase (V8) or papain. The activation with these proteinases required about 10 min to attain a maximal level, lasted 1/2-2 h and thereafter decreased gradually. Addition of an inhibitor of each proteinase resulted in an immediate cessation of any further changes in the enzymatic activity. The optimal pH of the proteinase-activated PDH was not affected. Proteinases increased the maximum velocity and the apparent Km values for pyruvate, but the Hill coefficients for pyruvate were unchanged. Proteinase-activated PDH was capable of associating two other component enzymes to produce large unit resembling the native complex. The Coomassie brilliant blue stained gels after SDS-PAGE showed that the PDH alpha subunit (41 kDa) was cleaved by trypsin or V8 into two major fragments (31 and 10 kDa), whereas PDH beta was unaffected. By amino-terminal sequence analyses of these fragments the trypsin cleavage sites were identified as Arg-273 and Arg-282 and the V8 cleavage sites were Glu-277 and Glu-280.
Koike
K
K
Department of Pathological Biochemistry, Nagasaki University School of Medicine, Japan.
Urata
Y
Y
Goto
S
S
D000595:Amino Acid Sequence; D000818:Animals; D004789:Enzyme Activation; D007700:Kinetics; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D010206:Papain / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010447:Peptide Hydrolases / Q000494:pharmacology*; D011487:Protein Conformation; D011768:Pyruvate Dehydrogenase Complex / Q000187:drug effects / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000494:pharmacology; D013552:Swine; D014357:Trypsin / Q000494:pharmacology
D010446:Peptide Fragments; D011768:Pyruvate Dehydrogenase Complex; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D010206:Papain