In vitro assembly of FAD, AMP, and the two subunits of electron-transferring flavoprotein: an important role of AMP related with the conformational change of the apoprotein.
Electron-transferring flavoprotein from pig kidney is composed of four non-covalently bound components: alpha and beta subunits, flavin adenine dinucleotide (FAD), and adenosine monophosphate (AMP). This paper reveals the pathway of assembly of the electron-transferring flavoprotein. The holoprotein can be formed by two different pathways. (i) alpha + beta <==> (alpha-beta)*, (alpha-beta)* + AMP <==> (alpha-beta-AMP)*, (alpha-beta-AMP)* <==> alpha-beta-AMP, alpha-beta-AMP + FAD <==> holoprotein. (ii) alpha + beta <==> alpha-beta, alpha-beta + FAD <==> alpha-beta-FAD, alpha-beta-FAD + AMP <==> holoprotein. Here the presence and absence of asterisks distinguish different conformations with the same composition. The monomeric forms of alpha and beta showed no significant binding with FAD and AMP. AMP and FAD associated with different heterodimer forms which were formed as a result of weak binding between alpha and beta. The binding of alpha + beta + AMP was much faster than that of alpha + beta + FAD because the rate of alpha + beta --> (alpha-beta)* was much faster than that of alpha + beta --> alpha-beta. The alpha-beta-AMP complex associated with FAD rapidly. As a result, the binding of FAD with the subunits is promoted by AMP. The alpha-beta-FAD complex associated with AMP much more slowly than the mixture of alpha and beta. Thus the AMP binding with the subunits is inhibited by the preceding FAD binding.
Sato
K
K
Department of Physiology, Kumamoto University School of Medicine, Honjo.
Nishina
Y
Y
Shiga
K
K
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000818:Animals; D004579:Electron Transport; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005420:Flavoproteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D013552:Swine
D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; D000249:Adenosine Monophosphate