ALTERING KINETIC MECHANISM AND ENZYME STABILITY BY MUTAGENESIS OF THE DIMER INTERFACE OF GLUTATHIONE-REDUCTASE

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