Carboxylic and polyethyleneimine-bound FAD derivatives. Synthesis, coenzymic properties, conformational and enzyme-coenzyme interaction studies

Alkylation at N‐1 of the FAD adenine ring with 3,4‐epoxybutanoic acid gave 1‐(2‐hydroxy‐3‐carboxypropyl)‐FAD. Dimroth rearrangement of the latter afforded N 6 ‐(2‐hydroxy‐3‐carboxypropyl)‐FAD. Coupling of the two carboxylic FAD derivatives to polyethyleneimine gave the corresponding soluble macromolecular FAD analogues. Fluorescence and circular dichroism determinations at neutral pH showed for the N‐1 carboxylic derivative an open conformation, while for the corresponding N 6 derivative a stacked one as for FAD. All the synthesized derivatives have been shown to reactivate Aspergillus niger glucose oxidase and hog kidney d ‐amino acid oxidase apoenzymes, affording holoenzymes whose activity relative to that of the corresponding natural holoenzymes ranged from 3 to 15% for the N‐1 derivatives and from 20 to 97% for the N 6 derivatives. Investigations by ultraviolet difference spectra on the interaction with apo‐ d ‐amino acid oxidase showed substantial similarities between the N 6 carboxylic derivative and FAD in the binding to the apoenzyme, while a completely different behaviour was observed for the N‐1 carboxylic derivative. The d ‐amino acid oxidase apoenzyme, the holoenzyme and the glutaraldehyde‐treated holoenzyme were entrapped each in cellulose triacetate fibres together with the polyethyleneimine N 6 derivative and beef liver catalase; the operational stability of the three recycling systems with dl ‐alanine as substrate was shown to be respectively 6, 5 and 12 times higher than the one found for the entrapped natural holoenzyme system.