Structural effects of a dimer interface mutation on catalytic activity of triosephosphate isomerase: the role of conserved residues and complementary mutations

Abstract

The active site of triosephosphate isomerase (TIM, EC: 5.3.1.1), a dimeric enzyme, lies very close to the subunit interface. Attempts to engineer monomeric enzymes have yielded well-folded proteins with dramatically reduced activity. The role of dimer interface residues in the stability and activity of the Plasmodium falciparum enzyme, PfTIM, has been probed by analysis of mutational effects at residue 74. The PfTIM triple mutant W11F/W168F/Y74W (Y74W^∗) has been shown to dissociate at low protein concentrations, and exhibits considerably reduced stability in the presence of denaturants, urea and guanidinium chloride. The Y74W^∗ mutant exhibits concentration-dependent activity, with an approximately 22-fold enhancement of k_cat over a concentration range of 2.5-40 μm, suggesting that dimerization is obligatory for enzyme activity. The Y74W^∗ mutant shows an approximately 20-fold reduction in activity compared to the control enzyme (PfTIM WT^∗, W11F/W168F). Careful inspection of the available crystal structures of the enzyme, together with 412 unique protein sequences, revealed the importance of conserved residues in the vicinity of the active site that serve to position the functional K12 residue. The network of key interactions spans the interacting subunits. The Y74W^∗ mutation can perturb orientations of the active site residues, due to steric clashes with proximal aromatic residues in PfTIM. The available crystal structures of the enzyme from Giardia lamblia, which contains a Trp residue at the structurally equivalent position, establishes the need for complementary mutations and maintenance of weak interactions in order to accommodate the bulky side chain and preserve active site integrity.