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See detailThe active site is the least stable structure in the unfolding pathway of a multidomain cold-adapted alpha-amylase
Siddiqui, K. S.; Feller, Georges ULg; D'Amico, Salvino ULg et al

in Journal of Bacteriology (2005), 187(17), 6197-6205

The cold-active alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30 degrees C ... [more ▼]

The cold-active alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30 degrees C) according to a two-state mechanism. Transverse urea gradient gel electrophoresis (TUG-GE) from 0 to 6.64 M was performed under various conditions of temperature (3 degrees C to 70 degrees C) and pH (7.5 to 10.4) in the absence or presence of Ca2+ and/or Tris (competitive inhibitor) to identify possible low-stability domains. Contrary to previous observations by strict thermal unfolding, two transitions were found at low temperature (12 degrees C). Within the duration of the TUG-GE, the structures undergoing the first transition showed slow interconversions between different conformations. By comparing the properties of the native enzyme and the N12R mutant, the active site was shown to be part of the least stable structure in the enzyme. The stability data supported a model of cooperative unfolding of structures forming the active site and independent unfolding of the other more stable protein domains. In light of these findings for AHA, it will be valuable to determine if active-site instability is a general feature of heat-labile enzymes from psychrophiles. Interestingly, the enzyme was also found to refold and rapidly regain activity after being heated at 70 degrees C for 1 h in 6.5 M urea. The study has identified. fundamental new properties of AHA and extended our understanding of structure/stability relationships of cold-adapted enzymes. [less ▲]

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See detailRole of disulfide bridges in the activity and stability of a cold-active alpha-amylase
Siddiqui, K. S.; Poljak, A.; Guilhaus, M. et al

in Journal of Bacteriology (2005), 187(17), 6206-6212

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis unfolds reversibly and cooperatively according to a two-state mechanism at 30 degrees C and unfolds reversibly and sequentially with two ... [more ▼]

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis unfolds reversibly and cooperatively according to a two-state mechanism at 30 degrees C and unfolds reversibly and sequentially with two transitions at temperatures below 12 degrees C. To examine the role of the four disulfide bridges in activity and conformational stability of the enzyme, the eight cysteine residues were reduced with beta-mercaptoethanol or chemically modified using iodoacetamide or iodoacetic acid. Matrix-assisted laser desorption-time of flight mass spectrometry analysis confirmed that all of the cysteines were modified. The iodoacetamide-modified enzyme reversibly folded/unfolded and retained approximately one-third of its activity. Removal of all disulfide bonds resulted in stabilization of the least stable region of the enzyme (including the active site), with a concomitant decrease in activity (increase in activation enthalpy). Disulfide bond removal had a greater impact on enzyme activity than on stability (particularly the active-site region). The functional role of the disulfide bridges appears to be to prevent the active site from developing ionic interactions. Overall, the study demonstrated that none of the four disulfide bonds are important in stabilizing the native structure of enzyme, and instead, they appear to promote a localized destabilization to preserve activity. [less ▲]

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