Kinetic and thermodynamic properties of the folding and assembly of formate dehydrogenase

The folding mechanism and stability of dimeric formate dehydrogenase from Candida methylica was analysed by exposure to denaturing agents and to heat. Equilibrium denaturation data yielded a dissociation constant of about 10 −13 M for assembly of the protein from unfolded chains and the kinetics of refolding and unfolding revealed that the overall process comprises two steps. In the first step a marginally stable folded monomeric state is formed at a rate ( k 1 ) of about 2 × 10 −3 s −1 (by deduction k −1 is about10 −4 s −1 ) and assembles into the active dimeric state with a bimolecular rate constant ( k 2 ) of about 2 × 10 4 M −1 s −1 . The rate of dissociation of the dimeric state in physiological conditions is extremely slow ( k −2 ∼ 3 × 10 −7 s −1 ).