CRYSTAL-STRUCTURE OF RECOMBINANT HUMAN TRIOSEPHOSPHATE ISOMERASE AT 2.8 ANGSTROM RESOLUTION - TRIOSEPHOSPHATE ISOMERASE-RELATED HUMAN GENETIC-DISORDERS AND COMPARISON WITH THE TRYPANOSOMAL ENZYME

The crystal structure of recombinant human triosephosphate isomerase ( hTIM) has been determined complexed with the transition-state analogue 2-phosphoglycolate at a resolution of 2.8 Angstrom. After refinement, the R-factor is 16.7% with good geometry. The asymmetric unit contain s 1 complete dimer of 53,000 Da, with only 1 of the subunits binding t he inhibitor. The so-called flexible loop, comprising residues 168-174 , is in its ''closed'' conformation in the subunit that binds the inhi bitor, and in the ''open'' conformation in the other subunit. The tips of the loop in these 2 conformations differ up to 7 Angstrom in posit ion. The RMS difference between hTIM and the enzyme of Trypanosoma bru cei, the causative agent of sleeping sickness, is 1.12 degrees for 487 C-alpha positions with 53% sequence identity. Significant sequence di fferences between the human and parasite enzymes occur at about 13 Ang strom from the phosphate binding site. The chicken and human enzymes h ave an RMS difference of 0.69 Angstrom for 484 equivalent residues and about 90% sequence identity. Complementary mutations ensure a great s imilarity in the packing of side chains in the core of the beta-barrel s of these 2 enzymes. Three point mutations in hTIM have been correlat ed with severe genetic disorders ranging from hemolytic disorder to ne uromuscular impairment. Knowledge of the structure of the human enzyme provides insight into the probable effect of 2 of these mutations, Gl u 104 to Asp and Phe 240 to Ile, on the enzyme. The third mutation rep orted to be responsible for a genetic disorder, Gly 122 to Arg, is how ever difficult to explain. This residue is far away from both catalyti c centers in the dimer, as well as from the dimer interface, and seems unlikely to affect stability or activity. Inspection of the 3-dimensi onal structure of trypanosomal triosephosphate isomerase, which has a methionine at position 122, only increased the mystery of the effects of the Gly to Arg mutation in the human enzyme.