Altering kinetic mechanism and enzyme stability by mutagenesis of the dimer interface of glutathione reductase

摘要: In wild-type glutathione reductase from Escherichia coli residues Val421 and Ala422 are located in an alpha-helix a densely packed hydrophobic region of the dimer interface, with their side chains against those Ala422' Val421' second subunit. A series mutant reductases was constructed which identities at positions 421 422 were changed. Mutations designed so as to present like charges (mutants Val421-->Glu:Ala422-->Glu Val421-->Lys:Ala422-->Lys) or opposite (mutant Val421-->Lys:Ala422-->Glu) across interface assess role electrostatic interactions stability. fourth (Val421-->His:Ala422-->His) also investigate effects introducing potentially protonatable bulky chain into crowded interface. all cases, active dimeric enzyme found be assembled but each protein thermally destabilized. detailed steady-state kinetic analysis indicated that no longer displayed Ping Pong behaviour associated exhibited what best described random bireactant ternary complex mechanism. This leads, depending on chosen substrate concentration, apparent sigmoidal, hyperbolic behaviour. These experiments, together others reported previously, indicate simple mutagenic changes regions distant site can lead dramatic switches