Unfolding-refolding transition of a hinge bending enzyme: horse muscle phosphoglycerate kinase induced by guanidine hydrochloride.

The research explores how horse muscle phosphoglycerate kinase, an enzyme, changes shape and function when treated with guanidine hydrochloride. It found that the two parts of the enzyme refold independently of each other, with different equilibrium constants, with the most optimal relating to the folding of the C-terminal domain containing all tryptophans.

Enzyme Study Subject

• The enzyme studied, horse muscle phosphoglycerate kinase, is part of a significant process in living cells as it involves in glycolysis, a fundamental energy producing process.
• This enzyme is often studied due to its ‘hinge-bending’ behaviour, meaning it changes shape during its operation and that change can be crucial for functionally.

Procedure and Methodology

• The researchers used the substance guanidine hydrochloride to induce a transition in the enzyme, where it unfolds and then refolds. This type of transition is crucial to the enzyme’s function and understanding this mechanism could provide insights into many biological processes.
• This process was studied under equilibrium conditions, meaning the researchers tried to maintain a consistent environment for the enzyme throughout the study.
• They used four different signals to measure the changes in the enzyme: fluorescence intensity at 336 nm, UV difference absorbance at 286 and 292 nm, ellipticity at 220 nm, and enzyme activity. These measures provide a detailed picture of how the enzyme changes during the unfolding-refolding transition.

Findings

• The research found that the unfolding-refolding transition process deviates from a two-state model. The two-state model is a simple way of thinking about enzyme transition, where it is either in a folded or unfolded state. The research’s findings suggested that there are intermediate states that also exist.
• Observed transition curves did not coincide, and the transition curve obtained from CD (Circular Dichroism) measurements was asymmetrical. Both of these findings suggest the existence of intermediates.
• Thermodynamic analysis and other data led the researchers to suggest that the two domains of the phosphoglycerate kinase refold independently of each other, each having a different equilibrium constant. An equilibrium constant refers to the ratio of the concentrations of the products and reactants in a reaction when it has reached equilibrium, in this case, in the refolding of the enzyme.
• The most favorable constant was found in the folding of the C-terminal domain of the enzyme. This region of the enzyme contains all of its tryptophan residues: tryptophan is an essential amino acid that is often involved in protein folding.