Glycerol-assisted Restorative Adjustment of Flavoenzyme Conformation Perturbed by Site-directed Mutagenesis
Year: 1997
Type: article
Abstract: 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.
Source: Journal of Biological Chemistry
Authors: Andrei A. Raibekas, Vincent Massey
Institution: University of Michigan
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
Award: G2869093499
OpenAlex ID: W2006468022