The purification and characterization of triose-phosphate isomerase from the psychrophilic bacterium Vibrio marinus (vTIM) is described. Crystal structures of the vTIM-sulfate complex and the vTIM-2-phosphoglycolate complex (at a 2.7-Å resolution) are also presented. The optimal growth temperature of Vibrio marinus is 15°C. Stability studies show that vTIM is an unstable protein with a half-life of only 10 min at 25°C. The vTIM sequence is most closely related to the sequence of Escherichia coli TIM (eTIM) (66% identity), and several unique structural features described for eTIM are also seen in vTIM, but eTIM is considerably more stable. The T d values of vTIM and eTIM, determined by calorimetric studies, are 41 and 54°C, respectively. Amino acid sequence comparison reveals that vTIM has an alanine in loop 8 (at position 238), whereas all other TIM sequences known to date have a serine. The vTIM mutant A238S was produced and characterized. Compared with wild type, the catalytic efficiency of the A238S mutant is somewhat reduced, and its stability is considerably increased.Triose-phosphate isomerase (TIM 1 ; EC 5.3.1.1) is a dimeric glycolytic enzyme formed by two identical subunits consisting of about 250 residues each. It catalyzes the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate (see Fig. 1). TIM has been the subject of extensive biophysical, enzymological, and computational studies. Its catalytic properties and mechanism have been studied in detail (1, 2). It has been established that TIM is a very efficient catalyst, since the reaction rates are diffusion-controlled. Neither cofactors nor metal ions are required in this reaction, and there is no evidence of allostery or cooperativity among the subunits.Structurally TIMs do form a well characterized family. To date, x-ray structures of TIM from seven different sources have been solved (wild type or/and in complex with substrate analogues): chicken (3), yeast (4), Trypanosoma brucei (5), Escherichia coli (6), human (7), Bacillus stearothermophilus (8), and Plasmodium falciparum (9). In addition, the structures have been determined of Leishmania mexicana TIM 2 and of Thermotoga maritima TIM. 3 Also, 45 amino acid TIM sequences from a wide variety of organisms have been determined and are available in the data bases.Each TIM monomer has a globular shape with two protruding loops. The globular part is formed by an 8-fold repeat of a ␤-strand, loop, ␣-helix, loop motif. The ␤␣ units fold up in a regular way, such that the ␤-strands (numbered ␤1 to ␤8) form an eight-stranded ␤-barrel surrounded by the eight ␣-helices (numbered ␣1 to ␣8) on the outside. This folding motif is also referred to as the TIM barrel motif. In TIM, the two protruding loops are after ␤-strand three and after ␤-strand six.The TIM barrel structural motif is found for a large number of other enzymes with widely different functions (10) that have little or no sequence homology. As such this stable framework with tolerant sequence variations has already been use...

In a Hungarian family with severe decrease in triosephosphate isomerase (TPI) activity, 2 germ line-identical but phenotypically differing compound heterozygote brothers inherited 2 independent (Phe240Leu and Glu145stop codon) mutations. The kinetic, thermodynamic, and associative properties of the recombinant human wild-type and Phe240Leu mutant enzymes were compared with those of TPIs in normal and deficient erythrocyte hemolysates. The specific activity of the recombinant mutant enzyme relative to the wild type was much higher (30%) than expected from the activity (3%) measured in hemolysates.