Mechanistic study of domain motions for the activation of enzyme I of the Escherichia coli phosphotransferase system : 대장균 인산기 전달 단백질 Enzyme I의 도메인 운동에 의한 활성화 메커니즘 연구

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농업생명과학대학 농생명공학부
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EIEICPEPdomain motiondimerizationautophosphorylation activitythermodynamics
학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2015. 2. 서정용.
Enzyme I (EI) functions only as a dimer as the first protein to initiate a series of phosphotransfer reactions in the bacterial phosphotransferase system (PTS), which catalyzes the sugar transport coupled with phosphorylation. PEP binding into EI induces not only large domain motion for autophosphorylation but also loop motion at the dimer interface, especially a loop β3α3, leading to tight dimerization. The structural link between the loop motion and the domain motions, however, was unclear. Here, we suggest a reason why only dimeric EI can be active by describing that the loop motion mechanically couples the tight dimerization and the catalytic domain motions. Mutation at the reside G356 perturbed the PEP induced structural change of the loop β3α3 and tight dimer state, resulting in a reduced autophosphorylation activity. The mutation did not affect the intrinsic affinity of EI for PEP, but significantly altered the catalytic domain motion, which amounted to a free energy loss of ~2 kcal mol-1. We further demonstrated that an impaired association between the catalytic domain and the PEP-binding domain was responsible for the altered domain motion. However, the reduced autophosphorylation activity was recovered by EI (H189A), which has a normal dimer interface, in the hetero dimer state. Taken together, we propose that the loop β3α3 at the dimer interface operates as an allosteric switch that connects PEP binding and domain motions to achieve the phosphoryl transfer reaction of the opposite subunit. Our findings would aid in the understanding of many puzzling aspects of bacterial physiology.
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Ph.D. / Sc.D._농생명공학부)
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