GLYCEROL-ASSISTED RESTORATIVE ADJUSTMENT OF FLAVOENZYME CONFORMATION PERTURBED BY SITE-DIRECTED MUTAGENESIS

The replacement of histidine 307 with leucine in pig kidney D amino ac id oxidase perturbs its active site conformation accompanied by dramat ic losses in protein-flavin interactions and enzymatic activity. Howev er, the negative effect of this mutation on the holoenzyme structure i s essentially eliminated in the presence of glycerol, resulting in up to 50% activity recovery and greater than 16-fold increase in the flav in affinity. Further analysis revealed that glycerol assists in the re arrangement of the protein toward its holoenzyme-like conformation tog ether with reduction in the solvent-accessible protein hydrophobic are a as demonstrated by limited proteolysis and use of affinity and hydro phobic probes. A substantial decrease in the protein-flavin interactio ns was demonstrated at a low temperature, but this reversible process was completely blocked in the presence of 40% glycerol. We suggest tha t the perturbation of the D-amino acid oxidase active site is due to t he nonpolar nature of the mutation whose negative impact on the holoen zyme structure can be overcome by glycerol-induced strengthening of pr otein internal hydrophobic interactions.