Organophosphorus hydrolase is a remarkably stable enzyme that unfolds through a homodimeric intermediate.
Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a homodimeric enzyme that catalyzes the hydrolysis of organophosphorus pesticides and nerve agents. We have analyzed the urea- and guanidinium chloride-induced equilibrium unfolding of OPH as monitored by far-ultraviolet circular dichroism and intrinsic tryptophan fluorescence. These spectral methods, which monitor primarily the disruption of protein secondary structure and tertiary structure, respectively, reveal biphasic unfolding transitions with evidence for an intermediate form of OPH. By investigating the protein concentration dependence of the unfolding curves, it is clear that the second transition involves dissociation of the monomeric polypeptide chains and that the intermediate is clearly dimeric. The dimeric intermediate form of OPH is devoid of enzymatic activity, yet clearly behaves as a partially folded, dimeric protein by gel filtration. Therefore, we propose an unfolding mechanism in which the native dimer converts to an inactive, well-populated dimeric intermediate which finally dissociates and completely unfolds to individual monomeric polypeptides. The denaturant-induced unfolding data are described well by a three-state mechanism with delta G for the interconversion between the native homodimer (N2) and the inactive dimeric intermediate (I2) of 4.3 kcal/mol while the overall standard state stability of the native homodimer relative to the unfolded monomers (2U) is more than 40 kcal/mol. Thus, OPH is a remarkably stable protein that folds through an inactive, dimeric intermediate and will serve as a good model system for investigating the energetics of protein association and folding in a system where we can clearly resolve these two steps.
Grimsley
J K
JK
Department of Biochemistry & Biophysics, Texas A&M University, College Station 77843-2128, USA.
Scholtz
J M
JM
Pace
C N
CN
Wild
J R
JR
D043303:Aryldialkylphosphatase; D002151:Calorimetry; D002850:Chromatography, Gel; D003196:Computer Graphics; D003198:Computer Simulation; D019281:Dimerization; D004926:Escherichia coli; D004950:Esterases / Q000737:chemistry* / Q000378:metabolism*; D019791:Guanidine; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D014508:Urea
D011994:Recombinant Proteins; D014508:Urea; D004950:Esterases; D043303:Aryldialkylphosphatase; D019791:Guanidine