Glycerol-assisted restorative adjustment of flavoenzyme conformation perturbed by site-directed mutagenesis.
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.
Raibekas
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AA
Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.
Massey
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V
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002384:Catalysis; D003605:D-Amino-Acid Oxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives / Q000378:metabolism; D005415:Flavins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D008958:Models, Molecular; D015335:Molecular Probes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation / Q000187:drug effects; D013499:Surface Properties; D013552:Swine
D000817:Anilino Naphthalenesulfonates; D005415:Flavins; D005456:Fluorescent Dyes; D015335:Molecular Probes; D005182:Flavin-Adenine Dinucleotide; D006639:Histidine; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); C077475:8-mercapto-FAD; D003605:D-Amino-Acid Oxidase; D005990:Glycerol