Refolding of pyridoxine-5'-P oxidase assisted by GroEL.
The unfolding of brain pyridoxine-5'-P oxidase by guanidinium chloride has been investigated at equilibrium. Circular dichroism, fluorescence spectroscopy and gel exclusion chromatography were used to monitor the unfolding process. The enzyme dissociates reversibly into monomers, but the fluorescence properties of the cofactor FMN are not restored upon dilution with potassium phosphate buffer (pH 7.4). Spontaneous refolding leads to 20% recovery of the catalytic activity. Addition of GroEL to the renaturing buffer accelerates the recovery of catalytic activity that approaches a level of 80% with respect to the native enzyme. The rate of recovery of catalytic activity assisted by GroEL parallels the rate of FMN fluorescence quenching, suggesting that structural rearrangements of the catalytic domain is the last step to take place in the refolding process.
Kwon
O S
OS
Department of Biochemistry, Kyungpook National University, Taegu 702-701, South Korea.
Churchich
J E
JE
D000818:Animals; D001921:Brain / Q000201:enzymology; D018834:Chaperonin 60 / Q000378:metabolism*; D002942:Circular Dichroism; D005486:Flavin Mononucleotide / Q000378:metabolism; D019791:Guanidine; D066298:In Vitro Techniques; D017510:Protein Folding; D011734:Pyridoxaminephosphate Oxidase / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine
D018834:Chaperonin 60; D005486:Flavin Mononucleotide; D011734:Pyridoxaminephosphate Oxidase; D019791:Guanidine