Thermostability of irreversible unfolding alpha-amylases analyzed by unfolding kinetics.
For most multidomain proteins the thermal unfolding transitions are accompanied by an irreversible step, often related to aggregation at elevated temperatures. As a consequence the analysis of thermostabilities in terms of equilibrium thermodynamics is not applicable, at least not if the irreversible process is fast with respect the structural unfolding transition. In a comparative study we investigated aggregation effects and unfolding kinetics for five homologous alpha-amylases, all from mesophilic sources but with rather different thermostabilities. The results indicate that for all enzymes the irreversible process is fast and the precedent unfolding transition is the rate-limiting step. In this case the kinetic barrier toward unfolding, as measured by unfolding rates as function of temperature, is the key feature in thermostability. The investigated enzymes exhibit activation energies (E(a)) between 208 and 364 kJmol(-1) and pronounced differences in the corresponding unfolding rates. The most thermostable alpha-amylase from Bacillus licheniformis (apparent transition temperature, T(1/2) approximately 100 degrees C) shows an unfolding rate which is four orders of magnitude smaller as compared with the alpha-amylase from pig pancreas (T(1/2) approximately 65 degrees C). Even with respect to two other alpha-amylases from Bacillus species (T(1/2) approximately 86 degrees C) the difference in unfolding rates is still two orders of magnitude.
Duy
Cihangir
C
Forschungszentrum Jülich, IBI-2, Biologische Strukturforschung, Jülich, Germany.
Fitter
Jörg
J
D000818:Animals; D001236:Aspergillus oryzae / Q000201:enzymology; D001407:Bacillus / Q000201:enzymology; D002942:Circular Dichroism; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D013552:Swine; D013816:Thermodynamics; D044366:Transition Temperature; D000516:alpha-Amylases / Q000737:chemistry* / Q000378:metabolism*
D000516:alpha-Amylases