Structural and Energetic Basis of Protein Kinetic Destabilization in Human Phosphoglycerate Kinase 1 Deficiency

Protein kinetic destabilization is a common feature of many
human genetic diseases. Human phosphoglycerate kinase 1 (PGK1) deficiency
is a rare genetic disease caused by mutations in the PGK1 protein, which often
shows reduced kinetic stability. In this work, we have performed an in-depth
characterization of the thermal stability of the wild type and four diseasecausing
mutants (I47N, L89P, E252A, and T378P) of human PGK1. PGK1
thermal denaturation is a process under kinetic control, and it is described well
by a two-state irreversible denaturation model. Kinetic analysis of differential
scanning calorimetry profiles shows that the disease-causing mutations
decrease PGK1 kinetic stability from ∼5-fold (E252A) to ∼100000-fold
(L89P) compared to that of wild-type PGK1, and in some cases, mutant
enzymes are denatured on a time scale of a few minutes at physiological
temperature. We show that changes in protein kinetic stability are associated
with large differences in enthalpic and entropic contributions to denaturation
free energy barriers. It is also shown that the denaturation transition state becomes more nativelike in terms of solvent exposure
as the protein is destabilized by mutations (Hammond effect). Unfolding experiments with urea further suggest a scenario in
which the thermodynamic stability of PGK1 at least partly determines its kinetic stability. ATP and ADP kinetically stabilize
PGK1 enzymes, and kinetic stabilization is nucleotide- and mutant-selective. Overall, our data provide insight into the structural
and energetic basis underlying the low kinetic stability displayed by some mutants causing human PGK1 deficiency, which may
have important implications for the development of native state kinetic stabilizers for the treatment of this disease.