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

Abstract

Edited by Miguel De la Rosa Keyword:Assembly mechanism Folding mechanism Formate dehydrogenase Candida methylica a b s t r a c tThe 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 À13 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 Â 10 À3 s À1 (by deduction k À1 is about10 À4 s À1 ) and assembles into the active dimeric state with a bimolecular rate con-. The rate of dissociation of the dimeric state in physiological conditions is extremely slow (k À2 $ 3 Â 10 À7 s À1 ).

Impact of Deep Eutectic Solvents on Kinetics and Folding Stability of Formate Dehydrogenase

“…The substantial usage of cofactors like NADH or NADPH in other industrially relevant enzyme systems incurs elevated expenses [21]. To mitigate these costs or even transform them into economic gains, extensive research has been dedicated to comprehending the cbFDH reaction in recent years [18,22,23]. Nevertheless, cbFDH encounters certain limitations in terms of its chemical, thermal, and long-term stability, along with the costly production of native cbFDHs and their limited enzymatic efficiency [24].…”

Protein Engineering Applications on Industrially Important Enzymes: Candida methylica FDH as a Case Study

“…According to this kinetic model, rate constant values yielded as yielded values of 1.9 ± 0.4 x10 -3 s -1 for the unimolecular folding step and 1.6 ± 0.5 x 10 4 M -1 s -1 for the bimolecular association of subunits (Ordu et al, 2009). The monomeric intermediate of cmFDH forms active dimers at a rate which is slower than expected for a process limited by subunit diffusion in solution.…”

“…The heat inactivation of cmFDH, as for most proteins, is not reversible and follows a firstorder decay. As a result of this, denaturation cannot be formally treated as an equilibrium system to which the orthodox analysis can be applied, rather it should be thought of as being defined by a temperature-sensitive rate constant for the irreversible step (Ordu et al, 2009). …”

“…The heat capacity change (ΔC p ) is -10.5 ± 1.8 kcal mol -1 K -1 ; a value that is dominated by the degree of desolvation of non-polar surface during the folding process (Ordu et al, 2009). …”