On the FAD-induced dimerization of apo-lipoamide dehydrogenase from Azotobacter vinelandii and Pseudomonas fluorescens. Kinetics of reconstitution.
The apoenzymes of lipoamide dehydrogenase from pig heart and from Pseudomonas fluorescens were prepared at pH 2.7 and pH 4.0, respectively, using a hydrophobic interaction chromatography procedure recently developed for lipoamide dehydrogenase from Azotobacter vinelandii and other flavoproteins [Van Berkel et al. (1988) Eur. J. Biochem. 178, 197-207]. The apoenzyme from pig heart, having 5% of residual activity, shows an equilibrium between the monomeric and dimeric species. Both the yield and the degree of reconstitution of dimeric holoenzyme is 75% of starting material under optimal conditions. The kinetics of reconstitution of pig heart apoenzyme differ slightly from that obtained with the apoenzyme prepared by acid ammonium sulfate precipitation at pH 1.5 [Kalse, J. F. and Veeger, C. (1968) Biochim. Biophys. Acta 159, 244-256]. The apoenzyme from P. fluorescens is in the monomeric state and shows negligible residual activity. The yield and degree of reconstitution of the dimeric holoenzyme is more than 90% of starting material. Reconstitution of the apoenzymes from A. vinelandii and P. fluorescens involves minimally a two-step sequential process. Initial flavin-binding results in regaining of full dichloroindophenol activity, quenching of tryptophan fluorescence and strong increase of FAD fluorescence polarization. In the second step, dimerization occurs as reflected by regain of lipoamide activity, strongly increased FAD fluorescence and increased hyperchroism of the visible absorption spectrum. The kinetics of FAD-induced dimerization are strongly dependent on the apoenzyme used. At 0 degrees C, the monomeric apoenzyme-FAD complex is either stabilized (P. fluorescens) or only transiently detectable (A. vinelandii). Dimerization of P. fluorescens enzyme is strongly stimulated in the presence of NADH.
van Berkel
W J
WJ
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Benen
J A
JA
Snoek
M C
MC
D000818:Animals; D001051:Apoenzymes / Q000378:metabolism*; D001395:Azotobacter / Q000201:enzymology*; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology; D011549:Pseudomonas / Q000201:enzymology*; D013057:Spectrum Analysis; D013552:Swine; D013696:Temperature
D001051:Apoenzymes; D005182:Flavin-Adenine Dinucleotide; D008058:Dihydrolipoamide Dehydrogenase