Role of disulfide bridges in the activity and stability of a cold-active alpha-amylase.
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.
Siddiqui
Khawar Sohail
KS
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales 2052, Australia.
Poljak
Anne
A
Guilhaus
Michael
M
Feller
Georges
G
D'Amico
Salvino
S
Gerday
Charles
C
Cavicchioli
Ricardo
R
D000064:Acclimatization; D003080:Cold Temperature; D003545:Cysteine; D004220:Disulfides / Q000378:metabolism*; D004795:Enzyme Stability; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D031161:Pseudoalteromonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics; D000516:alpha-Amylases / Q000737:chemistry* / Q000378:metabolism
D004220:Disulfides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000516:alpha-Amylases; D003545:Cysteine