Subunit associations in the mammalian pyruvate dehydrogenase complex. Structure and role of protein X and the pyruvate dehydrogenase component binding domain of the dihydrolipoyl transacetylase component.
We have further distinguished the structures and roles of the two lipoyl-bearing components of the pyruvate dehydrogenase complex, the dihydrolipoyl transacetylase (E2) component and the component designated as protein X. The amino acid sequences of the NH2-terminal regions of the lipoyl-bearing domain of the E2 component and protein X are different but related. The dihydrolipoyl dehydrogenase (E3) component but not the pyruvate dehydrogenase (E1) component protected protein X against proteolytic degradation by trypsin and protease Arg C. Protein X-specific polyclonal antibodies inhibit reconstitution of the overall reaction catalyzed by the complex (E2-X subcomplex recombined with the E1 and E3 components). The rate of development of this inhibition was reduced by pretreatment of E2-X subcomplex with the E3 component. These data strongly suggest the E3 component associates with protein X. The E1 component (an alpha 2 beta 2 tetramer), but not the E3 component, reduced trypsin cleavage of E2 subunits at 4 degrees C and altered the patterns of cleavage at 22 degrees C. At 22 degrees C a large (Mr congruent to 49,000) outer domain (E2LB) of the E2 component was produced. E2LB had the same NH2-terminal amino acid sequence as the smaller (Mr congruent to 38,000) lipoyl-bearing domain (E2L). E2LB, in contrast to E2L, interacted with both the E1 component and the beta subunit of the E1 component. Thus the E1 component is bound through an E1-binding domain that is located in E2 subunits between the inner domain and the outer, lipoyl-bearing domain.
Rahmatullah
M
M
Department of Biochemistry, Kansas State University, Manhattan 66506.
Gopalakrishnan
S
S
Andrews
P C
PC
Chang
C L
CL
Radke
G A
GA
Roche
T E
TE
D000123:Acetyltransferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology; D002417:Cattle; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D006801:Humans; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D051381:Rats
D046911:Macromolecular Substances; D011768:Pyruvate Dehydrogenase Complex; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase