Proteinase-catalyzed activation of porcine heart muscle pyruvate dehydrogenase and identification of its cleavage site

Abstract

Porcine heart muscle pyruvate dehydrogenase (PDH, EC 1.2.4.1) with subunit composition α2ß2 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 proteinase required about 10 min to attain a maximal level, lasted 12-2 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 PDHα subunit (41 kDa) was cleaved by trypsin or V8 into two major fragments (31 and 10 kDa), whereas PDHß 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.