Competitive Interaction of Component Enzymes with the Peripheral Subunit-Binding Domain of the Pyruvate Dehydrogenase Multienzyme Complex of Bacillus stearothermophilus: Kinetic Analysis Using Surface Plasmon Resonance Detection

摘要

The interactions of the peripheral enzymes (El, a pyruvate decarboxylase, and E3, dihydrolipoyl dehydrogenase) with the core component (E2, dihydrolipoyl acetyltransferase) of the pyruvate dehydrogenase (PDH) multienzyme complex of Bacillus stearothermophilus have been analyzed using a biosensor based on surface plasmon resonance detection. A recombinant di-domain (lipoyl domain plus peripheral subunit-binding domain) from E2 was attached to the biosensor chip by means of the pendant lipoyl group.The dissociation constant (K_d) for the complex between the peripheral subunit-binding domain and E3 (5.8 x 10~(-10) M) was found to be almost twice that for the complex with El (3.24 x 10~(-10) M). This was due to differences in the rate constants for dissociation (k_(diss)); these were 1.06 x 10~(-3) and 1,87 x 10~(-3) s~(-1) for the complexes with El and E3, respectively, whereas the rate constants for association (K_(ass)) were identical (3.26 x 10~6 M~(-1) s~(-1)). Separate studies using non-denaturing polyacryiamide gel electrophoresis confirmed the difference in affinity and demonstrated that El can rapidly displace E3 from an E3--di-domain complex and vice versa. The peripheral subunit-binding domain showed no detectable interaction with the Elαsubunit of El (α_2β_2) but exhibited a strong affinity for E1β (K_d = 8.5 x 10~(-9) M), confirming that the Elβ subunit is responsible for binding El to E2. These measurements introduce new features of potential importance inte the assembly and mechanism of the multienzyme complex.