Three hTIM mutants that provide new insights on why TIM is a dimer.

Mainfroid, Véronique and Terpstra, P and Beauregard, M and Frère, J M and Mande, S C and Hol, W G and Martial, J A and Goraj, K (1996) Three hTIM mutants that provide new insights on why TIM is a dimer. Journal of molecular biology, 257 (2). pp. 441-56. ISSN 0022-2836

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Human triosephosphate isomerase (hTIM), a dimeric enzyme, was altered by site-directed mutagenesis in order to determine whether it can be dissociated into monomers. Two hTIM mutants were produced, in which a glutamine residue was substituted for either Met14 or Arg98, both of which are interface residuces. These substitutions strongly interfere with TIM subunit association, since these mutant TIMs appear to exist as compact monomers in dynamic equilibrium with dimers. In kinetic studies, the M14Q mutant exhibits significant catalytic activity, while the R98Q enzyme is inactive. The M14Q enzyme is nevertheless much less active than unmutated hTIM. Moreover, its specific activity is concentration dependent, suggesting a dissociation process in which the monomers are inactive. In order to determine the conformational stability of the wild-type and mutant hTIMs, unfolding of all three enzymes was monitored by circular dichroism and tryptophan fluorescence spectroscopy. In each case, protein stability is concentration dependent, and the unfolding reaction is compatible with a two-state model involving the native dimer and unfolded monomers. The conformational stability of hTIM, as estimated according to this model, is 19.3 (+/-0.4) kcal/mol. The M14Q and R98Q replacements significantly reduce enzyme stability, since the free energies of unfolding are 13.8 and 13.5 (+/- 0.3) kcal/mol respectively, for the mutants, A third mutant, in which the M14Q and R98Q replacements are cumulated, behaves like a monomer. The stability of this mutant is not concentration-dependent, and the unfolding reaction is assigned to a transition from a folded monomer to an unfolded monomer. The conformational stability of this double mutant is estimated 2.5 (+/-0.1) kcal/mol. All these data combined suggest that TIM monomers are thermodynamically unstable. This might explain why TIM occurs only as a dimer.

Item Type: Article
Additional Information: Copyright of this article belongs to Elsevier Science.
Uncontrolled Keywords: (ba)-barrel; dimer; protein structure; stability; triosephosphate isomerase
Subjects: Q Science > QR Microbiology
Depositing User: Dr. K.P.S.Sengar
Date Deposited: 27 Jan 2012 15:29
Last Modified: 08 Jan 2015 10:24

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