Nonenzymatic cleavage of escherichia coli glutamine synthetase by dithiothreitol, $Fe^{3+}$, and $O_2$ = 디티오트레이톨·철 3가·산소계에 의한 대장균 글루타민 합성효소의 비효소적 절단

$\underline{\mbox{Escherichia}}$ $\underline{\mbox{coli}}$ glutamine synthetase is specifically fragmented by nonenzymatic metal-catalyzed oxidation system comprised of dithiothreitol, $Fe^{3+}$, and molecular oxygen. Several characteristics of the cleavage reaction were investigated. Optimum intrinsic or extrinsc reaction conditions on the $\underline{\mbox{E}}$. $\underline{\mbox{coli}}$ glutamine synthetase cleavage were 5 mM dithiothreitol, 0.1mM $Fe^{3+}$, pH 7.0, 37℃, and 4 hour of reaction time when the enzyme concentration was 0.06mM as subunit basis. Three dimensional structure of the enzyme was required to the specific nonenzymatic cleavage producing polypeptide fragments with defined molecular weights. The cleavage patterns were dramatically changed by addition of inherent ligands. This indicates that the cleavage is occurred at the active site of the enzyme. Three major polypeptides were isolated from the reaction mixture and their amino acid compositions were determined. Based on the molecular weight and amino acid composition, it was found that 47.8 kDa, 33.3 kDa, 28.9 kDa polypeptide belongs to C-, N-, and C-terminal of $\underline{\mbox{E}}$. $\underline{\mbox{coli}}$ glutamine synthetase, respectively. Amino acid sequences of the isolated polypeptide revealed that 29.0 kDa polypeptide was a peptide containing N-terminal of $\underline{\mbox{E}}$. $\underline{\mbox{coli}}$ glutamine synthetase and the N-terminals of other polypeptides were blocked. Molecular weights of 33.3 kDa and 28.9 kDa polypeptides were re-estimated as 29.0 kDa and 22.0 kDa respectively, with the molecular weight markers produced from hydroxylamine cleavage. From hydroxylamine cleavage of the fragments, major cleavage sites were identified as around amino acid position 31 and around 260 which are located on protein constructing active site of the enzyme. Probable fragmentation sites of 103 and 356 were predicted from investigation of amino terminally blocked peptide. But ma...
Byun, Si-Myungresearcher변시명researcher
Issue Date
61658/325007 / 000795230

학위논문(박사) - 한국과학기술원 : 생물공학과, 1991.2, [ ix, 110 p. ]

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