Structural and mutagenesis studies of leishmania triosephosphate isomerase: a point mutation can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power

The dimeric enzyme triosephosphate isomerase (TIM) has a very tight and rig id dimer interface. At this interface a critical hydrogen bond is formed be tween the main chain oxygen atom of the catalytic residue Lys13 and the com pletely buried side chain of Gln65 (of the same subunit), The sequence of L eishmania mexicana TIM, closely related to Trypanosoma brucei TIM (68 % seq uence identity), shows that this highly conserved glutamine has been replac ed by a glutamate, Therefore, the 1.8 Angstrom crystal structure of leishma nia TIM (at pH 5.9) was determined. The comparison with the structure of tr ypanosomal TIM shows no rearrangements in the vicinity of Glu65, suggesting that its side chain is protonated and is hydrogen bonded to the main chain oxygen of Lys13, Ionization of this glutamic acid side chain causes a pa-d ependent decrease in the thermal stability of leishmania TIM. The presence of this glutamate, also in its protonated state, disrupts to some extent th e conserved hydrogen bond network, as seen in all other TIMs, Restoration o f the hydrogen bonding network by its mutation to glutamine in the E65Q var iant of leishmania TIM results in much higher stability; for example, at pH 7, the apparent melting temperature increases by 26 degrees C (57 degrees C for leishmania TIM to 83 degrees C for the E65Q variant). This mutation d oes not affect the kinetic properties, showing that even point mutations ca n convert a mesophilic enzyme into a superstable enzyme without losing cata lytic power at the mesophilic temperature.