Relation between conformations and activities of lipoamide dehydrogenase. IV. Apoenzyme structure and flavin binding aspects

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1. 1. The apoenzyme of lipoamide dehydrogenase (NADH: lipoamide oxido-reductase, EC is thermolabile and urea-sensitive; the stability is also concentration-dependent. Upon prolonged incubation on ice the tertiary structure of concentrated apoenzyme (1 mg/ml) changes, though the flavin binding site is less affected. The apoenzyme partially withstands freezing for several weeks. 2. 2. Compared with that of the holoenzyme the protein fluorescence is increased in the apoenzyme; the excitation spectrum shows two maxima at 284 nm and 290 nm. Resolution of the difference fluorescence emission spectra showed a tyrosine contribution in the 305-nm region and non-polar tryptophan emission around 330 nm. 3. 3. The return of the lipoate activity is dependent on the apoenzyme concentration. Recombination systems with high apoenzyme concentrations (0.8 mg/ml) and excess of flavin develop a higher lipoate activity whilst the DCIP activity decreases faster than in the case of at lower apoenzyme concentrations (0.1 mg/ml). The dimerization is dependent on pH (7.2-7.5) and ionic strength (0.2 M). Urea, p-chloromercuribenzoate (PCMB) and l-cysteine disturb the recombination. 4. 4. Flavin derivatives are able to bind with the apoenzyme. Flavin-8-bromoadenine dinucleotide (FBAD) partially restores the DCIP activity (Kass= 1.5·104M-1). FMN is a competitive inhibitor of FAD binding and does not restore any activity. FMN derivatives generally show a non-competitive inhibition pattern, with respect to restoration of the DCIP activity. 3(N)-adducts of FMN exert very little inhibition. 2-Derivatives, such as 2-thio-FMN and 2-NH-phenyl-FMN, are less effectively bound, as also is tetra-hydro-FMN. The inhibitors induce, like FAD, conformational changes in the protein, resulting in a time-dependent increase of the binding forces between apoenzyme and flavin. 5. 5. Parts other than the isoalloxazine moiety of the flavin molecule, viz. the adenine part and pyrophosphate, are also involved in the FAD binding: NADH, NAD+, ADP, adenine, ATP and pyrophosphate inhibit the flavin binding. All nucleotides which inhibit the FAD binding affect the dimerization; only NAD+has a different behaviour in this respect since it promotes the return of the lipoate activity. © 1970.




Visser, J., & Veeger, C. (1970). Relation between conformations and activities of lipoamide dehydrogenase. IV. Apoenzyme structure and flavin binding aspects. BBA - Enzymology, 206(2), 224–241.

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