flavin-adenine-dinucleotide and 5-5--bis(8-(phenylamino)-1-naphthalenesulfonate)

The replacement of histidine 307 with leucine in pig kidney D-amino acid oxidase perturbs its active site conformation accompanied by dramatic losses in protein-flavin interactions and enzymatic activity. However, the negative effect of this mutation on the holoenzyme structure is essentially eliminated in the presence of glycerol, resulting in up to 50% activity recovery and greater than 16-fold increase in the flavin affinity. Further analysis revealed that glycerol assists in the rearrangement of the protein toward its holoenzyme-like conformation together with reduction in the solvent-accessible protein hydrophobic area as demonstrated by limited proteolysis and use of affinity and hydrophobic probes. A substantial decrease in the protein-flavin interactions was demonstrated at a low temperature, but this reversible process was completely blocked in the presence of 40% glycerol. We suggest that the perturbation of the D-amino acid oxidase active site is due to the nonpolar nature of the mutation whose negative impact on the holoenzyme structure can be overcome by glycerol-induced strengthening of protein internal hydrophobic interactions.

Topics: Anilino Naphthalenesulfonates; Animals; Binding, Competitive; Catalysis; D-Amino-Acid Oxidase; Flavin-Adenine Dinucleotide; Flavins; Fluorescent Dyes; Glycerol; Histidine; Kidney; Models, Molecular; Molecular Probes; Mutagenesis, Site-Directed; Protein Conformation; Surface Properties; Swine