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Role of Serine 310 in the Activity and Temperature Profile of Human Caspase-9
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Raheleh Shakeri    1, Mohadeseh Mahmoudian2 , Reza Khodarahmi3 , Soheila Mohammadi4 |
1- Assistant Professor, Department of Biological Science, Faculty of Science, University of Kurdistan, Sanandaj, Iran , r.shakeri@uok.ac.ir
2- MSc Student, Department of Biological Science, Faculty of Science, University of Kurdistan, Sanandaj, Iran
3- Professor, Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
4- Assistant Professor, Pharmaceutical Sciences Research Center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran. |
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Abstract: (1390 Views) |
Background and Aim: Caspase-9 is a key enzyme in the intrinsic pathway of apoptosis that its activity is regulated by various mechanisms such as phosphorylation. It has been reported that phosphorylation of serine 310 in murine caspase-9 prevents enzyme processing. The role of this residue in human caspase-9 activity in not clear. In this study we investigated the effect of negative charge on serine 310 in caspase-9 activity.
Materials and Methods: Considering that phosphorylation leads to negative charge on caspase-9, the codon of serine 310 in human capsase-9 was mutated to aspartate via quick change site-directed mutagenesis. Recombinant wild type and mutant caspase-9 were expressed in BL21(DE3) and purified by affinity chromatography. The temperature profile and activity of the mutant caspase-9 were assessed by chromogenic substrate of Ac-LEHD-pNA in vitro, and compared to those of wild enzyme. Student’s t test was used for data analysis.
Results: The results showed that kinetics parameters of S310D mutant and wild type caspase-9 were similar, but their temperature profiles were different. Comparison of S310D mutant enzyme and wild type caspase-9 showed that S310D mutant enzyme had higher activity at 37 oC and lower activity at 4, 15, 45 and 60 oC.
Conclusion: In our study, the negative charge on serine 310 in caspase-9 led to change in the profile temperature of the enzyme with no effect on kinetic parameters. |
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| Keywords: Caspase, Enzyme assay, Apoptosis, Mutagenesis |
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Full-Text [PDF 1084 kb]
(407 Downloads)
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Type of Study: Original Research |
Subject:
Biotechnology
Received: 2022/01/16 | Accepted: 2022/06/15 | Published: 2023/03/3
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