11. From the data table below, answer the following questions: a. What is the ro
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11. From the data table below, answer the following questions: a. What is the role of Ca2+ for the specific activity of CPK1? b. Why the double mutant s62A/S420A has lower specific activity? Table II. Phosphorylation activity for bacterially expressed and purified wild type and autophosphorylation site mutants of McCPK1-6x His fusion proteins assayed in the presence of histone H1 substrate McCPK1 Variant . Specific Activity x-Fold Ca2+ Activation Activitys versus wild-Type McCPK1 -Ca2 +Ca2 nu min mg Wild type S62A S420A S62A/5420A 0.54±0.010° 0.54±0.019 0.62 ± 0.011* 0.45 ± 0.016. 83.11±0.696" 89.81±2.37" 96.28 ± 1.255. 3.28 ± 0.028. 153.90 166.31 155.29 7.28 100 107.5 101.1 4.7Explanation / Answer
1. mcCPK1 is a salinity and dehydration stress-responsive calcium-dependent protein kinase (CDPK) in plants ,it is generally localized near the plasma membrane interact with myrismate in plasma membrane identified by green fluoresence protein.McCPK1 protein was catalytically active in presence a calcium cofactor.
Calcium is a ubiquitous and important second messenger in the signal transduction networks that plants use to respond to a wide variety of physiological stimuli. Cytosolic Ca2+fluctuations have been observed in response to a number of stimuli, including red light, abscisic acid , GA, drought , ionic stress, cold, heat shock.
So there is specific residues which interact with calcium ion (secreted highly during stress ) ,there is a EF hand motif on mcCPk1 which accept calcium ions .After binding to mcCPK1 ,calcium ion activates kinase activity of mcCPK1.
Then mcCPK1 activates cold ,high salt ,ABA ,Dark responsive promoters.so mcCPK IS REGULATED by calcium ions
2. Doubl substitution S62A andS420A mcCPK1 dramatically decreased the activity relative to the wild-type kinase. This suggests that simultaneous autophosphorylation of both Ser-62 and Ser-420 may be important for the activity of the enzyme. Ser-62 is located in the N-terminal variable domain. Ser-420 is located in the calmodulin-like domain between EF hand 1 and EF hand 2. Lack of autophosphorylation at these sites may limit the ability of the mutant kinase to undergo subtle conformational changes or impair the efficiency of Calcium ion bindin required for the activation of the enzyme relative to the wild-type kinase. Alternatively, the introduced double S62A/S420A substitution may have resulted in structural alterations in the kinase itself that could account for the reduced activity of the kinase.
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