Glycerol-induced development of catalytically active conformation of Crotalus adamanteus L-amino acid oxidase in vitro.
The reconstitutable apoprotein of Crotalus adamanteus L-amino acid oxidase was prepared using hydrophobic interaction chromatography. After reconstitution with flavin adenine dinucleotide, the resulting protein was inactive, with a perturbed conformation of the flavin binding site. Subsequently, a series of cosolvent-dependent compact intermediates was identified. The nearly complete activation of the reconstituted apoprotein and the restoration of its native flavin binding site was achieved in the presence of 50% glycerol. We provide evidence that in addition to a merely stabilizing effect of glycerol on native proteins, glycerol can also have a restorative effect on their compact equilibrium intermediates, and we suggest the hydrophobic effect as a dominating force in this in vitro-assisted restorative process.
Raibekas
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Department of Biological Chemistry, The University of Michigan Medical School, Ann Arbor 48109-0606, USA.
Massey
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D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D001051:Apoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D003435:Crotalid Venoms; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005990:Glycerol / Q000494:pharmacology*; D007700:Kinetics; D050830:L-Amino Acid Oxidase; D011487:Protein Conformation / Q000187:drug effects*; D013053:Spectrophotometry; D017819:Viperidae
D001051:Apoenzymes; D003435:Crotalid Venoms; D005182:Flavin-Adenine Dinucleotide; D000594:Amino Acid Oxidoreductases; D050830:L-Amino Acid Oxidase; D005990:Glycerol