3 HTIM MUTANTS THAT PROVIDE NEW INSIGHTS ON WHY TIM IS A DIMER

Human triosephosphate isomerase (hTIM), a dimeric enzyme, was altered by site-directed mutagenesis in order to determine whether it can be d issociated into monomers. Two hTIM mutants were produced, in which a g lutamine residue was substituted for either Met14 or Arg98, both of wh ich are interface residues. These substitutions strongly interfere wit h TIM subunit association, since these mutant TIMs appear to exist as compact monomers in dynamic equilibrium with dimers. In kinetic studie s, the M14Q mutant exhibits significant catalytic activity, while the R98Q enzyme is inactive. The M14Q enzyme is nevertheless much less act ive than unmutated hTIM. Moreover, its specific activity is concentrat ion dependent, suggesting a dissociation process in which the monomers are inactive. In order to determine the conformational stability of t he wild-type and mutant hTIMs, unfolding of all three enzymes was moni tored by circular dichroism and tryptophan fluorescence spectroscopy. In each case, protein stability is concentration dependent, and the un folding reaction is compatible with a two-state model involving the na tive dimer and unfolded monomers. The conformational stability of hTIM , as estimated according to this model, is 19.3(+/-0.4)kcal/mol. The M 14Q and R98Q replacements significantly reduce enzyme stability,since the free energies of unfolding are 13.8 and 13.5(+/-0.3)kcal/mol respe ctively, for the mutants. A third mutant, in which the M14Q and R98Q r eplacements are cumulated, behaves like a monomer. The stability of th is mutant is not concentration-dependent, and the unfolding reaction i s assigned to a transition from a folded monomer to an unfolded monome r. The conformational stability of this double mutant is estimated at 2.5(+/-0.1) kcal/mol. All these data combined suggest that TIM monomer s are thermodynamically unstable. This might explain why TIM occurs on ly as a dimer. (C) 1996 Academic Press Limited