Guanidine-induced equilibrium unfolding of a homo-hexameric enzyme 4-oxalocrotonate tautomerase (4-OT).
4-Oxalocrotonate tautomerase (4-OT) is a bacterial enzyme that is comprised of 6 identical 62 amino acid subunits. The 4-OT enzyme is an attractive model system in which to study the interrelationship between protein folding, subunit assembly, and catalytic function. Here we report on the GuHCl-induced equilibrium unfolding properties of wild-type 4-OT using catalytic activity measurements and using far-UV circular dichroism (CD) spectroscopy. We demonstrate that the unfolding of wild-type 4-OT in 50 mM phosphate buffers containing 6 M GuHCl is reversible at pHs 6.0, 7.4, and 8.5; and we find that there is both an enzyme concentration dependence and a pH dependence to the equilibrium unfolding properties of 4-OT. Our data suggests that the GuHCl-induced unfolding of 4-OT in 50 mM phosphate buffer at pH 8.5 can be modeled as a two-state process involving folded hexamer and unfolded monomer. On the basis of this model, we determined a free-energy value for the unfolding of 4-OT at pH 8.5 to be 68.7 +/- 3.2 kcal/mol under standard state conditions (1 M hexamer). In 50 mM phosphate buffers at pHs 6.0 and 7.4, only the catalytic activity denaturation curves are consistent with a two-state folding mechanism. At the lower pHs the far-UV-CD transitions are not well described by a two-state model. Our results at pHs 6.0 and 7.4 suggest that intermediate state(s) are populated in the equilibrium unfolding reaction at these lower pHs and that these intermediate state(s) have some helical content but no measurable catalytic activity.
Silinski
P
P
Department of Chemistry, Duke University, Durham, North Carolina 27708, USA.
Allingham
M J
MJ
Fitzgerald
M C
MC
D000595:Amino Acid Sequence; D002021:Buffers; D002384:Catalysis; D002942:Circular Dichroism; D019281:Dimerization; D019791:Guanidine; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D013011:Sorbic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D002021:Buffers; C087706:2-hydroxymuconic acid; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases; D019791:Guanidine; D013011:Sorbic Acid