Kinetics of the thermal inactivation and aggregate formation of rabbit muscle pyruvate kinase in the presence of trehalose.
In a previous study we found that 30-40% dimethylsulfoxide induces the active conformation of rabbit muscle pyruvate kinase. Because dimethylsulfoxide is known to perturb structure and function of many proteins, we have explored the effect of trehalose on the kinetics of thermal inactivation and stability of pyruvate kinase; this is because trehalose, in contrast to dimethyl sulfoxide, is totally excluded from the hydration shell of proteins. The results show that 600 mM trehalose inhibits the activity of pyruvate kinase by about 20% at 25 degrees C, however, trehalose protects pyruvate kinase from thermal inactivation at 60 degrees C, increases the Tm(app) of unfolding by 7.2 degrees C, induces a more compact state, and stabilizes its tetrameric structure. The inactivation process is irreversible due to the formation of protein aggregates. Trehalose diminishes the rate of formation of intermediates with propensity to aggregate, but does not affect the extent of aggregation. Remarkably, trehalose affects the aggregation process by inducing aggregates with amyloid-like characteristics.
Guerrero-Mendiola
Carlos
C
Departamento de Bioquímica, Facultad de Medicina, Apartado Postal 70-159, Universidad Nacional Autónoma de México, 04510 México, DF, Mexico.
Oria-Hernández
Jesús
J
Ramírez-Silva
Leticia
L
D000818:Animals; D004795:Enzyme Stability / Q000187:drug effects; D066298:In Vitro Techniques; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D020836:Protein Structure, Quaternary; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014199:Trehalose / Q000494:pharmacology*
D014199:Trehalose; D011770:Pyruvate Kinase