COMPETITIVE INTERACTION OF COMPONENT ENZYMES WITH THE PERIPHERAL SUBUNIT-BINDING DOMAIN OF THE PYRUVATE-DEHYDROGENASE MULTIENZYME COMPLEX OF BACILLUS-STEAROTHERMOPHILUS - KINETIC-ANALYSIS USING SURFACE-PLASMONRESONANCE DETECTION

The interactions of the peripheral enzymes (E1, a pyruvate decarboxyla se, and E3, dihydrolipoyl dehydrogenase) with the core component (E2, dihydrolipoyl acetyltransferase) of the pyruvate dehydrogenase (PDH) m ultienzyme complex of Bacillus stearothermophilus have been analyzed u sing a biosensor based on surface plasmon resonance detection. A recom binant di-domain (lipoyl domain plus peripheral subunit-binding domain ) from E2 was attached to the biosensor chip by means of the pendant l ipoyl group. The dissociation constant (K-d) for the complex between t he peripheral subunit-binding domain and E3 (5.8 x 10(-10) M) was foun d to be almost twice that for the complex with E1 (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 compl exes with E1 and E3, respectively, whereas the rate constants for asso ciation (k(ass)) were identical (3.26 x 10(6) M(-1) s(-1)). Separate s tudies using non-denaturing polyacrylamide gel electrophoresis confirm ed the difference in affinity and demonstrated that E1 can rapidly dis place E3 from an E3-di-domain complex and vice versa. The peripheral s ubunit-binding domain showed no detectable interaction with the E1 alp ha subunit of E1 (alpha(2) beta(2)) but exhibited a strong affinity fo r E1 beta (K-d = 8.5 x 10(-9) M), confirming that the E1 beta subunit is responsible for binding E1 to E2. These measurements introduce new features of potential importance into the assembly and mechanism of th e multienzyme complex.