Differences in the intersubunit contacts in triosephosphate isomerase from two closely related pathogenic trypanosomes.

The aligned amino acid sequences of TIM from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have a positional identity of 68%. The two enzymes have markedly similar catalytic properties. Agents that interact with their interface Cys inhibit TcTIM and TbTIM; and those TIMs that lack this Cys (such as human TIM) are largely or completely insensitive to these agents. The susceptibility of TcTIM to the agents is approximately 100 times higher than that of TbTIM. To ascertain the cause of this large difference, the crystal structure of TcTIM was solved at 1.83 A resolution. The two enzymes are very similar homodimers. In TcTIM and TbTIM their respective Cys, 15 or 14, forms part of the dimer interface. In both, the contacts of the Cys with residues of the other subunit are almost identical. Nevertheless, there are noteworthy differences between the two; the existence of glutamine 18 in TbTIM instead of glutamic acid in TcTIM at the beginning of helix 1 decreases the contacts between this portion of the protein and helix 3 of the other subunit. In addition, TcTIM has proline at position 24 in the first helix of the TIM barrel; this is absent in the other TIM. Pro24 disrupts the regular helix arrangement, making the pitch of this helix 1.2 A longer than in TbTIM. When Pro24 of TcTIM was substituted for Glu, the sensitivity of TcTIM to sulfhydryl reagents increased about fivefold, possibly as a consequence of an increase in the space between the first portion of helix 1 and helix 3 of the other subunit. Therefore, it may be concluded that the geometry of the latter region is central in the accessibility to agents that perturb the interface Cys. In human TIM this region is more compact.

[1]  Axel T. Brunger,et al.  Free R value: cross-validation in crystallography. , 1997 .

[2]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[3]  F. C. Hartman,et al.  Structure of yeast triosephosphate isomerase at 1.9-A resolution. , 1990, Biochemistry.

[4]  T. Nash,et al.  Complementation of an Escherichia coli glycolysis mutant by Giardia lamblia triosephosphate isomerase. , 1994, Experimental parasitology.

[5]  G. Petsko,et al.  Structure of chicken muscle triose phosphate isomerase determined crystallographically at 2.5Å resolution: using amino acid sequence data , 1975, Nature.

[6]  M. Noble,et al.  Comparison of the refined crystal structures of liganded and unliganded chicken, yeast and trypanosomal triosephosphate isomerase. , 1992, Journal of molecular biology.

[7]  G Vriend,et al.  Refined 1.83 A 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. , 1991, Journal of molecular biology.

[8]  T. A. Jones,et al.  Crystal structures of cellular retinoic acid binding proteins I and II in complex with all-trans-retinoic acid and a synthetic retinoid. , 1995, Structure.

[9]  M. L. Connolly.  Solvent-accessible surfaces of proteins and nucleic acids. , 1983, Science.

[10]  P. Focia,et al.  Hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi as a target for structure-based inhibitor design: crystallization and inhibition studies with purine analogs , 1997, Antimicrobial agents and chemotherapy.

[11]  A. Rojo-Domínguez,et al.  Species-specific inhibition of homologous enzymes by modification of nonconserved amino acids residues. The cysteine residues of triosephosphate isomerase. , 1996, European journal of biochemistry.

[12]  R. Read.  Improved Fourier Coefficients for Maps Using Phases from Partial Structures with Errors , 1986 .

[13]  P. Michels,et al.  Trypanosoma cruzi glycosomal glyceraldehyde‐3‐phosphate dehydrogenase: structure, catalytic mechanism and targeted inhibitor design , 1998, FEBS letters.

[14]  D. A. Fernández‐Velasco,et al.  Sequencing, expression and properties of triosephosphate isomerase from Entamoeba histolytica. , 1997, European journal of biochemistry.

[15]  J. Richardson,et al.  Amino acid preferences for specific locations at the ends of alpha helices. , 1988, Science.

[16]  J. Knowles,et al.  Enzyme catalysis: not different, just better , 1991, Nature.

[17]  A. Sun,et al.  Relationship between the catalytic center and the primary degradation site of triosephosphate isomerase: effects of active site modification and deamidation. , 1992, Archives of biochemistry and biophysics.

[18]  I. Becker,et al.  Cloning, expression, purification and characterization of triosephosphate isomerase from Trypanosoma cruzi. , 1997, European journal of biochemistry.

[19]  B. Shen,et al.  Human ornithine aminotransferase complexed with L-canaline and gabaculine: structural basis for substrate recognition. , 1997, Structure.

[20]  M Karplus,et al.  Structure of the triosephosphate isomerase-phosphoglycolohydroxamate complex: an analogue of the intermediate on the reaction pathway. , 1991, Biochemistry.

[21]  D. Tronrud,et al.  Knowledge-Based B-Factor Restraints for the Refinement of Proteins , 1996 .

[22]  C L Verlinde,et al.  Structure of the complex between trypanosomal triosephosphate isomerase and N‐hydroxy‐4‐phosphono‐butanamide: Binding at the active site despite an “open” flexible loop conformation , 1992, Protein science : a publication of the Protein Society.

[23]  M. Noble,et al.  Structure of triosephosphate isomerase from Escherichia coli determined at 2.6 A resolution. , 1993, Acta crystallographica. Section D, Biological crystallography.

[24]  A T Brünger,et al.  Protein hydration observed by X-ray diffraction. Solvation properties of penicillopepsin and neuraminidase crystal structures. , 1994, Journal of molecular biology.

[25]  B. Lee,et al.  The interpretation of protein structures: estimation of static accessibility. , 1971, Journal of molecular biology.

[26]  W. Kabsch,et al.  Crystal structure of the Trypanosoma cruzi trypanothione reductase·mepacrine complex , 1996, Proteins.

[27]  M. Noble,et al.  Structures of the “open” and “closed” state of trypanosomal triosephosphate isomerase, as observed in a new crystal form: Implications for the reaction mechanism , 1993, Proteins.

[28]  J. Knowles,et al.  Evolution of enzyme function and the development of catalytic efficiency. , 1976, Biochemistry.

[29]  R. A. Zubillaga,et al.  Using evolutionary changes to achieve species-specific inhibition of enzyme action--studies with triosephosphate isomerase. , 1995, Chemistry & biology.

[30]  J. Martial,et al.  Crystal structure of recombinant human triosephosphate isomerase at 2.8 Å resolution. Triosephosphate isomerase‐related human genetic disorders and comparison with the trypanosomal enzyme , 1994, Protein science : a publication of the Protein Society.

[31]  T. Borchert,et al.  An interface point‐mutation variant of triosephosphate isomerase is compactly folded and monomeric at low protein concentrations , 1995, FEBS letters.

[32]  W G Hol,et al.  Three hTIM mutants that provide new insights on why TIM is a dimer. , 1996, Journal of molecular biology.

[33]  C. Verlinde,et al.  Protein crystallography and infectious diseases , 1994, Protein science : a publication of the Protein Society.

[34]  L. Wyns,et al.  Triose-phosphate isomerase (TIM) of the psychrophilic bacterium Vibrio marinus. Kinetic and structural properties. , 1998, The Journal of biological chemistry.

[35]  E. Saavedra-Lira,et al.  Sulfhydryl reagent susceptibility in proteins with high sequence similarity--triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi and Leishmania mexicana. , 1998, European journal of biochemistry.

[36]  A. Brünger,et al.  Torsion angle dynamics: Reduced variable conformational sampling enhances crystallographic structure refinement , 1994, Proteins.

[37]  R J Fletterick,et al.  The crystal structure of cruzain: a therapeutic target for Chagas' disease. , 1995, Journal of molecular biology.

[38]  J. Knowles,et al.  Stabilization of a reaction intermediate as a catalytic device: definition of the functional role of the flexible loop in triosephosphate isomerase. , 1990, Biochemistry.

[39]  J. Knowles,et al.  Triosephosphate isomerase: energetics of the reaction catalyzed by the yeast enzyme expressed in Escherichia coli. , 1988, Biochemistry.

[40]  A. Fairlamb,et al.  The crystal structure of trypanothione reductase from the human pathogen Trypanosoma cruzi at 2.3 Å resolution , 1996, Protein science : a publication of the Protein Society.

[41]  H. Balaram,et al.  Triosephosphate isomerase from Plasmodium falciparum: the crystal structure provides insights into antimalarial drug design. , 1997, Structure.

[42]  J. Martial,et al.  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 , 1995, Protein science : a publication of the Protein Society.