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Transient kinetic studies reveal isomerization steps along the kinetic pathway of T hermus thermophilus 3‐isopropylmalate dehydrogenase
Author(s) -
Gráczer Éva,
Lionne Corinne,
Závodszky Péter,
Chaloin Laurent,
Vas Mária
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12191
Subject(s) - isomerization , kinetic energy , chemistry , kinetics , thermus thermophilus , transient (computer programming) , dehydrogenase , enzyme , biophysics , biochemistry , biology , catalysis , physics , computer science , escherichia coli , quantum mechanics , gene , operating system
To identify the rate‐limiting step(s) of the 3‐isopropylmalate dehydrogenase‐catalysed reaction, time courses of NADH production were followed by stopped flow ( SF ) and quenched flow ( QF ). The steady state k cat and K m values did not vary between enzyme concentrations of 0.1 and 20 μ m . A burst phase of NADH formation was shown by QF , indicating that the rate‐limiting step occurs after the redox step. The kinetics of protein conformational change(s) induced by the complex of 3‐isopropylmalate with Mg 2+ were followed by using the fluorescence resonance energy transfer signal between protein tryptophan(s) and the bound NADH . A reaction scheme was proposed by incorporating the rate constant of a fast protein conformational change (possibly domain closure) derived from the separately recorded time‐dependent formation of the fluorescence resonance energy transfer signal. The rate‐limiting step seems to be another slower conformational change (domain opening) that allows product release.