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.
The interactions of the peripheral enzymes (E1, 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 (Kd) 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 E1 (3.24 x 10(-10) M). This was due to differences in the rate constants for dissociation (kdiss); these were 1.06 x 10(-3) and 1.87 x 10(-3) s-1 for the complexes with E1 and E3, respectively, whereas the rate constants for association (kass) were identical (3.26 x 10(6) M-1 s-1). Separate studies using non-denaturing polyacrylamide gel electrophoresis confirmed the difference in affinity and demonstrated that E1 can rapidly displace E3 from an E3-di-domain complex and vice versa. The peripheral subunit-binding domain showed no detectable interaction with the E1 alpha subunit of E1 (alpha 2 beta 2) but exhibited a strong affinity for E1 beta (Kd = 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 the multienzyme complex.
Lessard
I A
IA
Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, United Kingdom.
Fuller
C
C
Perham
R N
RN
D001665:Binding Sites; D001667:Binding, Competitive; D015374:Biosensing Techniques; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D011487:Protein Conformation; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013057:Spectrum Analysis
D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins