TRIOSE-PHOSPHATE-ISOMERASE (TIM) OF THE PSYCHROPHILIC BACTERIUM VIBRIO-MARINUS - KINETIC AND STRUCTURAL-PROPERTIES

The purification and characterization of triose-phosphate isomerase fr om the psychrophilic bacterium Vibrio marinus (vTIM) is described. Cry stal structures of the vTIM-sulfate complex and the vTIM-2-phosphoglyc olate complex (at a 2.7-Angstrom resolution) are also presented, The o ptimal growth temperature of Vibrio marinus is 15 degrees C. Stability studies show that vTIM is an unstable protein with a half-life of onl y 10 min at 25 degrees C. The vTIM sequence is most closely related to the sequence of Escherichia coli TIM (eTIM) (66% identity), and sever al unique structural features described for eTIM are also seen in vTIM , but eTIM is considerably more stable. The T-d values of vTIM and eTI M, determined by calorimetric studies, are 41 and 54 degrees C, respec tively. Amino acid sequence comparison reveals that vTIM has an alanin e in loop 8 (at position 238), whereas all other TIM sequences known t o date have a serine. The vTIM mutant A238S was produced and character ized, Compared with wild type, the catalytic efficiency of the A238S m utant is somewhat reduced, and its stability is considerably increased .