TY  - JOUR
AU  - Fitter, J.
AU  - Haber-Pohlmeier, S.
TI  - Structural Stability and Unfolding Properties of Thermostable bacterial alpha-amylases: A Comparative Study on Homologous Enzymes
JO  - Biochemistry
VL  - 43
SN  - 0006-2960
CY  - Columbus, Ohio
PB  - American Chemical Society
M1  - PreJuSER-42202
SP  - 9589 - 9599
PY  - 2004
N1  - Record converted from VDB: 12.11.2012
AB  - In a comparative investigation on two thermostable alpha-amylases [Bacillus amyloliquefaciens (BAA), T(m) = 86 degrees C and Bacillus licheniformis (BLA), T(m) = 101 degrees C], we studied thermal and guanidine hydrochloride (GndHCl)-induced unfolding using fluorescence and CD spectroscopy, as well as dynamic light scattering. Depletion of calcium from specific ion-binding sites in the protein structures reduces the melting temperature tremendously for both alpha-amylases. The reduction is nearly the same for both enzymes, namely, in the order of 50 degrees C. Thus, the difference in thermostability between BLA and BAA (DeltaT(m) approximately 15 degrees C) is related to intrinsic properties of the respective protein structures themselves and is not related to the strength of ion binding. The thermal unfolding of both proteins is characterized by a full disappearance of secondary structure elements and by a concurrent expansion of the 3D structure. GndHCl-induced unfolding also yields a fully vanishing secondary structure but with more expanded 3D structures. Both alpha-amylases remain much more compact upon thermal unfolding as compared to the fully unfolded state induced by chemical denaturants. Such rather compact thermal unfolded structures lower the conformational entropy change during the unfolding transition, which principally can contribute to an increased thermal stability. Structural flexibilities of both enzymes, as measured with tryptophan fluorescence quenching, are almost identical for both enzymes in the native states, as well as in the unfolded states. Furthermore, we do not observe any difference in the temperature dependence of the structural flexibilities between BLA and BAA. These results indicate that conformational dynamics on the time scale of our studies seem not to be related to thermal stability or to thermal adaptation.
KW  - Bacillus: enzymology
KW  - Bacterial Proteins: chemistry
KW  - Enzyme Stability
KW  - Guanidine: chemistry
KW  - Kinetics
KW  - Light
KW  - Protein Denaturation
KW  - Protein Folding
KW  - Scattering, Radiation
KW  - Sequence Homology, Amino Acid
KW  - Spectrometry, Fluorescence
KW  - Temperature
KW  - Thermodynamics
KW  - alpha-Amylases: chemistry
KW  - Bacterial Proteins (NLM Chemicals)
KW  - Guanidine (NLM Chemicals)
KW  - alpha-Amylases (NLM Chemicals)
KW  - J (WoSType)
LB  - PUB:(DE-HGF)16
C6  - pmid:15274613
UR  - <Go to ISI:>//WOS:000222965100002
DO  - DOI:10.1021/bi0493362
UR  - https://juser.fz-juelich.de/record/42202
ER  -