The folding pathway of glycosomal triosephosphate isomerase: structural insights into equilibrium intermediates.

The guanidine hydrochloride-induced conformational transitions of glycosomal triosephosphate isomerase (TIM) were monitored with functional, spectroscopic, and hydrodynamic measurements. The equilibrium folding pathway was found to include two intermediates (N(2) ↔I(2) ↔2M↔2U). According to this model, the conformational stability parameters of TIM are as follows: ΔG(I2-N2) = 5.5 ± 0.6, ΔG(2M-I2) =19.6 ± 1.6, and ΔG(2U-2M) = 14.7 ± 3.1 kcal mol(-1) . The I(2) state is compact (α(SR) = 0.8); it is able to bind 8-anilinonaphthalene-1-sulfonic acid ANS and it is composed of ∼45% of α-helix and tertiary structure content compared with the native enzyme; however, it is unable to bind the transition-state analog 2-phosphoglycolate. Conversely, the 2M state lacks detectable tertiary contacts, possesses ∼10% of the native α-helical content, is significantly expanded (α(SR) = 0.2), and has low affinity for ANS. We studied the effect of mutating cysteine residues on the structure and stability of I(2) and 2M. Three mutants were made: C39A, C126A, and C39A/C126A. The replacement of C39, which is located at β(2) , was found to be neutral. The I(2) -C126A state, however, was prone to aggregation and exhibited an emission maximum that was 3-nm red-shifted compared with the I(2) -wild type, indicating solvent exposure of W90 at β(4) . Our results suggest that the I(2) state comprises the (βα)(1-4) β(5) module in which the conserved C126 residue located at β(5) defines the boundary of the folded segment. We propose a folding pathway that highlights the remarkable thermodynamic stability of this glycosomal enzyme.