Mass Spectrometry of Hydrogen/Deuterium Exchange of <i>Escherichia coli</i> Dihydrofolate Reductase: Effects of Loop Mutations

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  • Yamamoto Tatsuya
    Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University
  • Izumi Shunsuke
    Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University
  • Ohmae Eiji
    Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University
  • Gekko Kunihiko
    Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University

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  • Mass Spectrometry on Hydrogen/Deuterium Exchange of Escherichia coli Dihydrofolate Reductase: Effects of Loop Mutations
  • Mass Spectrometry of Hydrogen/Deuterium Exchange of Escherichia coli Dihydrofolate Reductase: Effects of Loop Mutations

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To address the effects of single amino acid substitutions on the structural fluctuation of Escherichia coli dihydrofolate reductase (DHFR), hydrogen/deuterium exchange kinetics were investigated at 15°C with wild-type and mutant DHFRs at Gly 67 (six mutants) and Gly 121 (eight mutants) located in two flexible loops, by means of electrospray ionization mass spectrometry. These mutations induced significant changes in the first-order rate constant of proton exchange, kex (0.10-0.27min-1), the number of fast-exchangeable protons, ΔMo (164-222 Da), and the number of protons protected from exchange, ΔM∞ (15-56 Da), relative to the corresponding values for the wild-type enzyme (kex=0.18min-1, ΔMo=164 Da, and ΔM∞=50.5 Da). These kinetic parameters were strongly correlated with the volume of introduced amino acids, but partly correlated with adiabatic compressibility (volume fluctuation), stability, and enzymatic activity. These results indicate that the local structure change due to a single amino acid substitution in loop regions is dramatically magnified to affect the structural fluctuation of the whole DHFR molecule, resulting in complicated changes in its stability and function.

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