Investigation of enzymatic properties and structural changes of cysteine substituted mutants of human cytochrome c
1
Issued Date
2023
Copyright Date
2023
Resource Type
Language
eng
File Type
application/pdf
No. of Pages/File Size
xvi, 154 leaves : ill.
Access Rights
open access
Rights
ผลงานนี้เป็นลิขสิทธิ์ของมหาวิทยาลัยมหิดล ขอสงวนไว้สำหรับเพื่อการศึกษาเท่านั้น ต้องอ้างอิงแหล่งที่มา ห้ามดัดแปลงเนื้อหา และห้ามนำไปใช้เพื่อการค้า
Rights Holder(s)
Mahidol University
Bibliographic Citation
Thesis (Ph.D. (Chemistry))--Mahidol University, 2023
Suggested Citation
Sasiprapa Samsri Investigation of enzymatic properties and structural changes of cysteine substituted mutants of human cytochrome c. Thesis (Ph.D. (Chemistry))--Mahidol University, 2023. Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/115314
Title
Investigation of enzymatic properties and structural changes of cysteine substituted mutants of human cytochrome c
Author(s)
Abstract
Cytochrome c (Cytc) is well-known as an electron transporter in mitochondria. Additional function of Cytc is an initiator in the apoptotic pathway by acting as a peroxidase. This process involves conformational changes in Cytc, including structural adjustments at the Ω-loop segments, which facilitate the opening of the heme crevice to the active site. Mutations in Cytc at Ω-loops have been studied for their impact on peroxidase activity. In this work, to examine how the Ω-loops of human Cytochrome c (huCytc) involve in the peroxidase activity, the researcher prepared four mutants located in three different regions of the Ω-loops, i.e., T28C and G34C (proximal Ω-loop), A50C (central Ω-loop), and P76C (distal Ω-loop). The biophysical properties of these mutants were then compared to the wild-type (WT) huCytc. In addition, the flexibility at the sites of the mutated cysteines was analyzed using site-directed spin-labeling electron spin resonance (SDSL-ESR). Moreover, the data from molecular dynamics (MD) simulations were incorporated to observe detailed structural and dynamics caused by the mutations. The results of these structural and dynamic analyses were then compared to the modulation of peroxidase activity. According to Michaelis-Menten kinetic model, it was found that the peroxidase activity was increased from WT by ~13, ~2.6, and ~2 folds for P76C, G34C, and A50C, respectively. Meanwhile T28C mutant and WT were found to be comparable. Interestingly, the ESR data showed that the most restricted site was at T28C while P76C showed the highest flexibility of the loop at this region of huCytc, which is consistent with the peroxidase activity results. Additionally, the MD results suggested the important changes in P76C mutant occurred due to the more opening of cavity where it is related to active site resulting in enhancing peroxidase activity. Moreover, the G34C mutant caused local destabilization and flexibility at the proximal Ω-loop. Besides the mutation at distal Ω-loop, the mutation on proximal Ω-loop (G34C) also modulate the structure to increasing peroxidase activity. The investigation of conformational changes of huCytc and its related peroxidase activity would contribute to the benefit on its application in vitro and guide a way to understand huCytc to the role of apoptosis in the environments similar to those in cell.
Degree Name
Doctor of Philosophy
Degree Level
Doctoral degree
Degree Department
Faculty of Science
Degree Discipline
Chemistry
Degree Grantor(s)
Mahidol University