pH-dependent relationship between thermodynamic and kinetic stability in the denaturation of human phosphoglycerate kinase 1.
Human phosphoglycerate kinase 1 (hPGK1) is a glycolytic enzyme essential for ATP synthesis, and it is implicated in different pathological conditions such as inherited diseases, oncogenesis and activation of drugs for cancer and viral treatments. Particularly, mutations in hPGK1 cause human PGK1 deficiency, a rate metabolic conformational disease. We have recently found that most of these mutations cause protein kinetic destabilization by significant changes in the structure/energetics of the transition state for irreversible denaturation. In this work, we explore the relationships between protein conformation, thermodynamic and kinetic stability in hPGK1 by performing comprehensive analyses in a wide pH range (2.5-8). hPGK1 remains in a native conformation at pH 5-8, but undergoes a conformational transition to a molten globule-like state at acidic pH. Interestingly, hPGK1 kinetic stability remains essentially constant at pH 6-8, but is significantly reduced when pH is decreased from 6 to 5. We found that this decrease in kinetic stability is caused by significant changes in the energetic/structural balance of the denaturation transition state, which diverge from those found for disease-causing mutations. We also show that protein kinetic destabilization by acidic pH is strongly linked to lower thermodynamic stability, while in disease-causing mutations seems to be linked to lower unfolding cooperativity. These results highlight the plasticity of the hPGK1 denaturation mechanism that responds differently to changes in pH and in disease-causing mutations. New insight is presented into the different factors contributing to hPGK1 thermodynamic and kinetic stability and the role of denaturation mechanisms in hPGK1 deficiency.
Department of Physical Chemistry, Faculty of Sciences, University of Granada, Av./Fuentenueva s/n, E-18071 Granada, Spain. Electronic address: angelpey@ugr.es.
Angel L
AL
Pey
D004795:Enzyme Stability; D040181:Genetic Diseases, X-Linked / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008661:Metabolism, Inborn Errors / Q000201:enzymology; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000172:deficiency / Q000378:metabolism*; D011489:Protein Denaturation; D012997:Solvents / Q000737:chemistry; D013816:Thermodynamics
D012997:Solvents; C577282:PGK1 protein, human; D010735:Phosphoglycerate Kinase
Phosphoglycerate kinase; Protein denaturation mechanism; Protein kinetic stability; Protein thermodynamic stability