A proposed mechanism for the thermal denaturation of a recombinant Bacillus halmapalus alpha-amylase--the effect of calcium ions.
The thermal stability of a recombinant alpha-amylase from Bacillus halmapalus alpha-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). This alpha-amylase is homologous to other Bacillus alpha-amylases where crystallographic studies have identified the existence of three calcium binding sites in the structure. Denaturation of BHA is irreversible with a T(m) of approximately 89 degrees C and DSC thermograms can be described using a one-step irreversible model. A 5 degrees C increase in T(m) in the presence of 10-fold excess CaCl(2) was observed. However, a concomitant increase in the tendency to aggregate was also observed. The presence of 30-40-fold excess calcium chelator (ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis[beta-aminoethyl ether] N,N,N',N'-tetraacetic acid (EGTA)) results in a large destabilization of BHA, corresponding to about 40 degrees C lower T(m) as determined by both CD and DSC. Ten-fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which probably originate from different populations of BHA/calcium complexes. Combined interpretation of these observations and structural information on homologous alpha-amylases forms the basis for a suggested mechanism underlying the inactivation mechanism of BHA. The mechanism includes irreversible thermal denaturation of different BHA/calcium complexes and the calcium binding equilibria. Furthermore, the model accounts for a temperature-induced reversible structural change associated with calcium binding.
Department of Life Sciences and Chemistry, Roskilde University, P.O. Box 260, 1-Universitetsvej, DK-4000 Roskilde, Denmark.
Anders D
AD
Nielsen
Marc L
ML
Pusey
Claus C
CC
Fuglsang
Peter
P
Westh
D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D002122:Calcium Chloride / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D002152:Calorimetry, Differential Scanning; D002614:Chelating Agents / Q000737:chemistry / Q000494:pharmacology; D002942:Circular Dichroism; D004492:Edetic Acid / Q000737:chemistry / Q000494:pharmacology; D004533:Egtazic Acid / Q000737:chemistry / Q000494:pharmacology; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics
D002614:Chelating Agents; D011994:Recombinant Proteins; D004533:Egtazic Acid; D004492:Edetic Acid; D000516:alpha-Amylases; D002122:Calcium Chloride