Poster: Enzymology

Abs # 253: Is pyruvate dehydrogenase kinase regulated by dihydrolipoyl acetyltransferase?

Presenter:	Tovar Mendez, Alejandro , tovarmendeza@missouri.edu
Authors	Tovar Mendez, Alejandro  (A)   Miernyk, Jan A. (B) (A)  Randall, Douglas D. (A)
Affiliations:	(A): Biochemistry Dept., University of Missouri-Columbia (B): Plant Genetics Research Unit, USDA-ARS, University of Missouri

The mitochondrial pyruvate dehydrogenase complex (mtPDC) consists of multiple copies of three noncovalently associated enzymes: pyruvate dehydrogenase (E1), dihydrolipoyl acetyltransferase (E2), and dihydrolipoyl dehydrogenase (E3). The E2 component forms the 60mer core of the complex to which the other components bind. In addition, mtPDC includes the E1-kinase (PDK) and P-E1-phosphatase regulatory enzymes. Overall PDC activity is controlled by multi-site phosphorylation of the E1a subunit. The mtPDK from animal cells binds to the lipoyl domain from E2. Binding of PDK to E2 activates the kinase, and this activation is increased by high NADH/NAD and acetyl-CoA/CoA ratios. Details of this activation are unclear. It is not known if or how PDK binds to plant mtPDC. Additionally, neither NADH nor acetyl-CoA activate phosphorylation of E1 by plant PDK. Some preliminary data suggest that plant PDK binds to the E2 lipoyl domain. Additionally, the activity of recombinant Arabidopsis thaliana PDK (AtPDK), using kinase-depleted PDC from pea seedlings as the substrate, was 10-fold higher than with recombinant Arabidopsis thaliana E1 was the substrate. We are studying the interactions among E1, E2, and PDK in vitro with a system reconstituted entirely from recombinant A. thaliana proteins. The A. thaliana genome includes a single PDK gene, and three genes encoding mitochondrial E2 proteins (two mono-lipoyl forms of E2, plus one of the more typical di-lipoyl forms). The structural, catalytic, and regulatory roles of each E2 isoform is under evaluation.