Abstract
Four catalytic amino acids at triosephosphate isomerase (TIM) are highly conserved: N11, K13, H95, and E167. Asparagine 11 is the last of these to be characterized in mutagenesis studies. The ND2 side chain atom of N11 is hydrogen bonded to the O-1 hydroxyl of enzyme-bound dihydroxyacetone phosphate (DHAP), and it sits in an extended chain of hydrogen-bonded side chains that includes T75' from the second subunit. The N11A variants of wild-type TIM from Trypanosoma brucei brucei (TbbTIM) and Leishmania mexicana (LmTIM) undergo dissociation from the dimer to monomer under our assay conditions. Values of K(as) = 8 × 10(3) and 1 × 10(6) M(-1), respectively, were determined for the conversion of monomeric N11A TbbTIM and LmTIM into their homodimers. The N11A substitution at the variant of LmTIM previously stabilized by the E65Q substitution gives the N11A/E65Q variant that is stable to dissociation under our assay conditions. The X-ray crystal structure of N11A/E65Q LmTIM shows an active site that is essentially superimposable on that for wild-type TbbTIM, which also has a glutamine at position 65. A comparison of the kinetic parameters for E65Q LmTIM and N11A/E65Q LmTIM-catalyzed reactions of (R)-glyceraldehyde 3-phosphate (GAP) and (DHAP) shows that the N11A substitution results in a (13-14)-fold decrease in k(cat)/K(m) for substrate isomerization and a similar decrease in k(cat) for DHAP but only a 2-fold decrease in k(cat) for GAP.