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

Abstract

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 N6-(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 N6 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 N6 derivatives. Investigations by ultraviolet difference spectra on the interaction with apo-d-amino acid oxidase showed substantial similarities between the N6 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 N6 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.