PMID:15115773
| Citation |
Yamamoto, T, Izumi, S, Ohmae, E and Gekko, K (2004) Mass spectrometry of hydrogen/deuterium exchange of Escherichia coli dihydrofolate reductase: effects of loop mutations. J. Biochem. 135:487-94 |
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| Abstract |
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 degrees C with wild-type and mutant DHFRs at Gly67 (six mutants) and Gly121 (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, k(ex) (0.10-0.27 min(-1)), the number of fast-exchangeable protons, Delta M(o) (164-222 Da), and the number of protons protected from exchange, Delta M(infinity) (15-56 Da), relative to the corresponding values for the wild-type enzyme (k(ex) = 0.18 min(-1), Delta M(o) = 164 Da, and Delta M(infinity) = 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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| Keywords |
Algorithms; Amino Acids/chemistry; Deuterium Exchange Measurement/methods; Deuterium Oxide/chemistry; Escherichia coli/enzymology; Kinetics; Molecular Weight; Mutagenesis, Site-Directed; Point Mutation; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Spectrometry, Mass, Electrospray Ionization; Tetrahydrofolate Dehydrogenase/chemistry; Tetrahydrofolate Dehydrogenase/genetics; Thermodynamics |
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