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The Structural and Functional Analysis of the Amino Acid of the Subunit Interface of Glutathione S-transferase

Author: WangCaiLing
Tutor: YangHaiLing
School: Beijing Forestry University
Course: Biochemistry and Molecular Biology
Keywords: Glutathione S-transferases mutagenesis Enzyme assays Structure
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 29
Quote: 0
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Abstract


TaGSTU4 was taken as study material and constructed site-directed mutagenesis of these conserved residues. The recombinant proteins were overexpressed in E.coli and studied the enzyme analysis and structure.The main results were as follows:1、Structural investigations of a TaGSTU4 showed two subunit interactions:a hydrogen bond between the Tyr93 and Pro65 from another subunit of the dimer; and two salt bridges between residues Glu78 and side chains of Arg95 and Arg99 in the opposite subunit. These residues are conserved in all tau class GSTs in plant.2、The recombinant mutants were constructed using the site-mutant PCR, including E78D, E78A, Y93A, Y93F, R95A, R95K, R99A and R99L. All the recombinant proteins were identified soluble or insoluble in E.coli.3、After purification, all soluble recombinant proteins showed a single band in SDS-PAGE, and the subunit Mw of the recombinant proteins should be 26kDa. Matrix assisted laser desorption ionization time-of-flight/mass spectrometry (MALDI-TOF/MS) confirmed the subunit Mw of the mutants and wild type enzymes was about 26kDa. Native-PAGE and gel filtration chromatography showed the molecular weights of the mutants and wild type enzymes were between 43kDa and 66kDa. These results strongly indicated that the recombinant proteins were a homodimer of 26kDa.4、Substrate specificity activities showed:(1) All mutants lost activity to substrates NBD-CL and NBC. (2)A dramatic reduction in activity towards the substrate CDNB was found for the mutants E78D,Y93F and R95K.(3)The mutants E78D,Y93F, R95K and the wild type showed similar activities towards the substrate cumene hydroperoxide.In contrast, the mutants Y93A,R95A and R99L did not show any activity towards this substrate.5、Kinetic activities showed the mutants Y93F、E78D and R95K showed lower catalytic efficiency towards substrates GSH and CDNB, compared with the wild type, suggesting that these residues played key roles in enzymes catalytic functions.6、ANS is used to monitor the appearance and disappearance of hydrophobic patches or surfaces on the proteins. The mutants E78D, Y93A, Y93F, R95A and R95K showed marked increases in ANS fluorescence intensity, compared with the wild type enzyme, indicating enhanced exposure of hydrophobic surface.All the above results indicated that the hydrogen bond between the Tyr93 and Pro65 had an important effect on enzymatic catalytic, thermal and structural stability. Salt bridges played important roles in the structural stability and catalytic activities.

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