Unfolding of 4-aminobutyrate aminotransferase equilibrium and kinetic studies.
The unfolding of pig liver 4-aminobutyrate aminotransferase by urea has been investigated at equilibrium. The overall process was reversible as judged from the recovery of catalytic activity after dilution of urea-treated samples. Unfolding of the enzyme was monitored by circular dichroism and fluorescence spectroscopy. The steepness of the fluorescence and CD changes between 2 and 8 M urea, and the lack of any discernible plateau suggests that unfolding of the protein is a cooperative process. The unfolding of 4-aminobutyrate aminotransferase as a function of urea concentration was monitored by fluorescence measurements of the tryptophanyl residues. The kinetic results indicate that the aminotransferase unfolds in a single kinetic phase. Unfolded 4-aminobutyrate aminotransferase recovers immediately its catalytic activity upon dilution with buffers of neutral pH. Based on equilibrium and kinetic results, a two-state model for the unfolding of the aminotransferase is proposed.
Pineda
T
T
Department of Biochemistry, University of Tennessee, Knoxville 37916.
Churchich
J E
JE
D000612:4-Aminobutyrate Transaminase / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002021:Buffers; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013552:Swine
D002021:Buffers; D000612:4-Aminobutyrate Transaminase