Mass Spectrometry on Hydrogen/Deuterium Exchange of Dihydrofolate Reductase: Effects of Ligand Binding
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概要
- 論文の詳細を見る
To address the effects of ligand binding on the structural fluctuations of Escherichia coli dihydrofolate reductase (DHFR), the hydrogen/deuterium (H/D) exchange kinetics of its binary and ternary complexes formed with various ligands (folate, dihydrofolate, tetrahydrofolate, NADPH, NADP+, and methotrexate) were examined using electrospray ionization mass spectrometry. The kinetic parameters of HID exchange reactions, which consisted of two phases with fast and slow rates, were sensitively influenced by ligand binding, indicating that changes in the structural fluctuation of the DHFR molecule are associated with the alternating binding and release of the cofactor and substrate. No additivity was observed in the kinetic parameters between a ternary complex and its constitutive binary complexes, indicating that ligand binding cooperatively affects the structural fluctuation of the DHFR molecule via longrange interactions. The local H/D exchange profile of pepsin digestion fragments was determined by matrix-assisted laser desorption/ionization mass spectrometry, and the helix and loop regions that appear to participate in substrate binding, largely fluctuating in the apo-form, are dominantly influenced by ligand binding. These results demonstrate that the structural fluctuation of kinetic intermediates plays an important role in enzyme function, and that mass spectrometry on H/D exchange coupled with ligand binding and protease digestion provide new insight into the structure-fluctuation-function relationship of enzymes.
- 社団法人 日本生化学会の論文
著者
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GEKKO Kunihiko
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University
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Gekko Kunihiko
Department Of Food Science And Technology Faculty Of Agriculture Nagoya University
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Izumi Shunsuke
Department Of Chemistry Facalty Of Science Hiroshima University
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Yamamoto Tatsuya
Department Of Applied Chemistry Graduate School Of Engineering Hiroshima University
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