Role of Disulfide Bridges in the Activity and Stability of a Cold-Active α-Amylase

Abstract

The cold-adapted ␣-amylase from Pseudoalteromonas haloplanktis unfolds reversibly and cooperatively according to a two-state mechanism at 30°C and unfolds reversibly and sequentially with two transitions at temperatures below 12°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 ␤-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.

The Folding Process of Acylphosphatase from Escherichia coli is Remarkably Accelerated by the Presence of a Disulfide Bond

“…These data are in agreement with previously published results describing a mutational variant of EcoAcP in which only one of the two cysteine residues is substituted by an alanine residue. 26 Except for a few cases, 39 it has been generally reported that disulfide bonds are responsible for a stabilization of the overall three-dimensional structure of proteins of different sizes, regardless of their structural organization. [40][41][42][43][44][45][46][47] It is clear that the native disulfide bond of EcoAcP also has a marked influence on the folding process of this protein.…”

A novel thermostable α-1,3-glucanase from <i>Streptomyces thermodiastaticus</i> HF 3-3

“…Until now, no report has demonstrated the involvement of thiol groups in catalytic mechanism of a-1,3-glucanase or the other glycosidases. On the other hand, many studies have suggested the role of thiol groups in maintaining the architecture of the active site (Krajewska, 2008;Siddiqui et al, 2005). It is possible that a-1,3-glucanase HF65 might contain a disulfide bond that contributes to activity as well as thermal stability.…”

Structural insights into cold inactivation of tryptophanase and cold adaptation of subtilisin S41

“…[36][37][38][39] They offer economic benefits through energy savings, in skipping the expensive heating steps, as well as minimizing the chemical reactions or degradations of products that occur at higher temperatures. 36,37,40 Consequently, considerable efforts are directed towards identifying the specific structural features responsible for the activity and stability of these enzymes (reviewed 37,[40][41][42][43][44] ). However, thus far, only a limited number of cold-adapted enzymes have been characterized structurally using X-ray crystallography.…”