[en] 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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Mainfroid, Véronique; Université de Liège - ULiège
Terpstra, Peter; University of Groningen
Beauregard, Marc; University of Prince Edward Island
Frère, Jean-Marie ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Mande, Shekhar C; University of Washington
Hol, Wim G; University of Washington
Martial, Joseph ; Université de Liège - ULiège > Département des sciences de la vie > GIGA-R : Biologie et génétique moléculaire
Goraj, Karine; Université de Liège - ULiège
Language :
English
Title :
Three hTIM mutants that provide new insights on why TIM is a dimer
Alber, T., Banner, D W., Bloomer, A. C., Petsko, G. A., Phillips, D. C., Rivers, P. S. & Wilson, I. A. (1981). On the three-dimensional structure and catalytic mechanism of triosephosphate isomerase. Phil. Trans. Roy. Soc. ser. B, 293, 159-171.
Banner, D. W., Bloomer A. C., Petsko, G. A., Phillips, D. C., Pogson, C. I., Wilson, I. A., Corran, P H., Furth, A. J., Milman, J. D., Offord, R. E., Priddle, J. D. & Waley, S. G. (1975). Structure of chicken muscle triosephosphate isomerase determined crystallographically at 2.5 A resolution using amino acid sequence data. Nature, 255, 609-614.
Borchert, T. V., Pratt, K., Zeelen, J. Ph., Callens, M., Noble, M. E. M., Opperdoes, F. R., Michels, P. A. M. & Wierenga, R. K. (1993a). Overexpression of trypanosomal triosephosphate isomerase in Escherichia coli and characterization of a dimer-interface mutant. Eur. J. Biochem. 211, 703-710.
Borchert, T. V., Abagyan, R., Radha Kishan, K. V., Zeelen, J. Ph. & Wierenga, R. K. (1993b). Structure of an engineered monomeric triosephosphate isomerase, monoTIM: the correct modelling of an eight-residue loop. Structure, 1, 205-213.
Brändén, C. I. (1991). The most frequently occuring folding motif in proteins-the TIM barrel. Curr. Opin. Struct. Biol. 1, 978-983.
Brandts, J. F. (1964). The thermodynamics of protein denaturation. J. Am. Chem. Soc. 86, 4291-4301.
Burstein, E. A. (1976). Luminescence of Protein Chromophores. vol. 6, Model Studies. Science and Technology Results. Biophysics, VINITI, Moscow.
Burstein, E. A. (1977). Intrinsic Protein Luminescence. vol. 7, Origin and Applications. Science and Technology Results. Biophysics, VINITI, Moscow.
Casal, J. I., Ahern, T. J., Davenport, R. C., Petsko, G. A. & Klibanov, A. M. (1987). Subunit interface of triosephosphate isomerase: site-directed mutagenesis and characterization of the altered enzyme. Biochemistry, 26, 1258-1264.
Suite (1994). Programs for protein crystallography Acta Crystallog. sect. D, 50, 760-763.
Chan, W. W. C. & Mawer, H. M. (1972). Protein subunits-preparation and properties of active subunits of aldolase bound to a matrix. Arch. Biochem. Biophys. 149, 136-145.
Chan, W. W. C., Mort, J. S., Chong, D. K. K. & Macdonald, P. D. M. (1973a). Protein subunits-kinetic evidence for the presence of active subunits during the renaturation of muscle aldolase. J. Biol. Chem. 248, 2778-2784.
Chan, W. W. C., Schutt, H. & Brand, K. (1973b). Active subunits of transaldolase bound to Sepharose. Eur. J. Biochem. 40, 533-541.
Delboni, L. F., Mande, S. C., Rentier-Delrue, F., Mainfroid, V., Turley S., Vellieux, F. M. D., Martial, J. A. & Hol, W. G. J. (1995). Crystal structure of recombinant triosephosphate isomerase from Bacillus stearothermophilus. An analysis of potential thermostability factors in six isomerases with known three dimensional structures points to the importance of hydrophobic interactions. Protein Sci. 4, 2594-2604.
De Moerlooze, L., Struman, I., Renard, A. & Martial, J. A. (1992). Stabilization of T7-promoter-based pARHS expression vectors using the parB locus. Gene, 119, 91-93.
Eftink, M. R., Helton, K. J., Beavers, A. & Ramsay, G. D. (1994). The unfolding of trp aporepressor as a function of pH: evidence for an unfolding intermediate. Biochemistry, 33, 10220-10228.
Eisenberg, D. & McLachlan, A. D. (1986). Solvation energy in protein folding and binding. Nature, 319, 199-203.
Erickson, H. P. (1989). Co-operativity in protein-protein association. The structure and stability of the actin filament. J. Mol. Biol. 206, 465-474.
Farber, G. K. & Petsko, G. A. (1990). The evolution of α/β barrel enzymes. Trends Biochem. Sci. 15, 228-234.
Gally J. A. & Edelman, G. M. (1964). Effects of conformation and environment on the fluorescence of proteins and polypeptides. Biopolymers, Symp. 1, 367-381.
Goraj, K., Renard, A. & Martial, J. A. (1990). Synthesis, purification and initial structural characterization of octarellin, a de novo polypeptide modelled on the α/β-barrel proteins. Protein Eng. 3, 259-266.
Herold, M. & Kirschner, K. (1990). Reversible dissociation and unfolding of aspartate aminotransferase from Escherichia coli: characterization of a monomeric intermediate. Biochemistry, 29, 1907-1913.
Horton, N. & Lewis, M. (1992). Calculation of the free energy of association for protein complexes. Protein Sci. 1, 169-181.
Jaenicke, R. (1987). Folding and association of proteins. Prog. Biophys. Mol. Biol. 49, 117-237.
Jones, D. H., McMillan, A. J. & Fersht, A. R. (1985). Reversible dissociation of dimeric tyrosyl-tRNA synthetase by mutagenesis at the subunit interface. Biochemistry, 24, 5852-5857.
Jones, S. & Thornton, J. M. (1995). Protein-protein interactions: a review of protein dimer structures. Prog. Biophys. Mol. Biol. 63, 31-65.
Knowles, J. R. (1991). Enzyme catalysis: not different, just better. Nature, 350, 121-124.
Komives, E. A., Chang, L. C., Lolis, E., Tilton, R. F., Petsko, G. A. & Knowles, J. R. (1991). Electrophilic catalysis in triosephosphate isomerase: The role of histidinegs. Biochemistry, 30, 3011-3019.
Kunkel, T. A. (1985). Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl Acad. Sci. USA, 82, 488-492.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature, 227, 680-685.
Leatherbarrow, R. J. (1987). Enzfitter: a non-linear regression data analysis program for the IBMPC/ P52. Elsevier Biosoft, Cambridge, UK.
Lee, E. H., Soper, T. S., Mural, R. J., Stringer, C. D. & Hartman, F. C. (1987). An intersubunit interaction at the active site of D-ribulose-1,5-bisphosphate carboxylase/oxygenase as revealed by cross-linking and site-directed mutagenesis. Biochemistry, 26, 4599-4604.
Lodi, P. J., Chang, L. C., Knowles, J. R. & Komives, E. A. (1994). Triosephosphate isomerase requires a positively charged active site: the role of lysine-12. Biochemistry, 33, 2809-2814.
Lolis, E., Alber, T., Davenport, R. C., Rose, D., Hartman, F. C. & Petsko, G. A. (1990). Structure of yeast triosephosphate isomerase at 1.9 Å resolution. Biochemistry, 29, 6609-6618.
Mande, S. C., Mainfroid, V., Kalk, K. H., Goraj, K., Martial, J. A. & Hol, W. G. J. (1994). Crystal structure of recombinant human triosephosphate isomerase at 2.8 Å resolution. Protein Sci. 3, 810-821.
Misset, O. & Opperdoes, F. R. (1984). Simultaneous purification of hexokinase, class-I fructose-bisphosphate aldolase, triosephosphate isomerase and phosphoglycerate kinase from Trypanosoma brucei. Eur. J. Biochem. 144, 475-483.
Mossing, M. C. & Sauer, R. T. (1990). Stable, monomeric variants of λcro obtained by insertion of a designed β-hairpin sequence. Science, 250, 1712-1715.
Neet, K. E. & Timm, D. E. (1994). Conformational stability of dimeric proteins: quantitative studies by equilibrium denaturation. Protein Sci. 3, 2167-2174.
Noble, M. E. M., Zeelen, J. P., Wierenga, R. K., Mainfroid, V., Goraj, K., Gohimont, A. C. & Martial, J. A. (1993). Structure of triosephosphate isomerase from E. coli determined at 2.6 Å resolution. Acta Crystallog. sect. D, 49, 403-417.
Nordoff, A., Bücheler, U. S., Werner, D. & Schirmer, R. H. (1993). Folding of the four domains and dimerization are impaired by the Gly446-Glu exchange in human glutathione reductase. Implications for the design of antiparasitic drugs. Biochemistry, 32, 4060-4066.
Pace, C. N. (1990). Conformational stability of globular proteins. Trends Biochem. Sci. 15, 14-17.
Pace, C. N., Shirley, B. A. & Thomson, J. A. (1989). Measuring the conformational stability of a protein. In Protein Structure: A Practical Approach (Creighton, T. E., ed.), pp. 311-330, IRL Press, Oxford.
Privalov, P. L. & Khechinashvili, N, N. (1974). A thermodynamic approach to the problem of stabilization of globular protein structure: a calorimetric study J. Mol. Biol. 86, 665-684.
Rieder, S. V. & Rose, I. A. (1959). The mechanism of the triosephosphate isomerase reaction. J. Biol. Chem. 234, 1007-1010.
Schein, C. H. (1989). Production of soluble recombinant proteins in bacteria. Biotechnology, 7, 1141-1148.
Studier, F. W. & Moffatt, B. A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113-130.
Vriend, G. (1990). WHAT IF: a molecular modeling and drug design program. J. Mol. Graph. 8, 52-56.
Waley, S. G. (1973). Refolding of triosephosphate isomerase. Biochem. J. 135, 165-172.
Wierenga, R. K., Kalk, K. H. & Hol, W. G. J. (1987). Structure determination of the glycosomal triosephosphate isomerase from Trypanosoma brucei brucei at 2.4 Å resolution. J. Mol. Biol. 198, 109-121.
Wierenga, R. K., Noble, M. E. M., Vriend, G., Nauche, S. & Hol, W. G. J. (1991). Refined 1.83 Å structure of trypanosomal triosephosphate isomerase crystallized in the presence of 2.4 M ammonium sulphate. A comparison with the structure of the trypanosomal triosephosphate isomerase-glycerol-3-phosphate complex. J. Mol. Biol. 220, 995-1015.
Wierenga, R. K., Noble, M. E. M. & Davenport, R. C. (1992). Comparison of the refined crystal structures of liganded and unliganded chicken, yeast and trypanosomal triosephosphate isomerase. J. Mol. Biol. 224, 1115-1126.
Wolfenden, R. (1969). Transition state analogues for enzyme catalysis. Nature, 233, 704-705.
