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 K(m) 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.