Mapping the uncharted water channel of dihydrodipicolinate synthase : a proposed mechanism of allostery.
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
Dihydrodipicolinate synthase catalyses the condensation reaction of pyruvate and aspartate semialdehyde in the first committed step of the diaminopimelic acid pathway that synthesizes lysine. Dihydrodipicolinate synthase is allosterically inhibited by lysine.
This study hypothesises that a water channel that connects the lysine binding site and the active site is critical for allosteric inhibition by lysine. When lysine binds to the allosteric site, it acts as a lid for the water channel, blocking the ability to shuttle protons to the active site from bulk solvent, slowing the reaction. To mimic the lysine in the allosteric site, two substituted enzymes were created with a point substitution at the 48 position, which is positioned in the water channel. The substitutions inserted are phenylalanine and tryptophan (DHDPS-S48F and DHDPS-S48W), since their bulky sidechains should block the water channel.
Kinetic studies show both substituted enzymes had a slower rate than the native. The DHDPS-S48F enzyme had a similar rate to the native with lysine, consistent with our hypothesis. The DHDPS-S48W enzyme had a much slower rate than the other two, and when lysine was in the assay there was no change to the rate. Therefore, either lysine did not bind, or it had no effect on the enzyme.
The substituted enzymes were crystallised with substrates and lysine to ensure the only interference was the water channel being blocked, and to identify any changes in the structure as a result of ligand binding. The catalytic sites of both substituted enzymes were unaffected by the substitutions. There was slight variation in Y107 in both enzymes, more so in the tryptophan substitution, but this was only a minor change. The allosteric site was also unaffected by the substitutions, yet lysine was not present in the DHDPS-S48W, despite extensive soaking. This could have been due to Y106 being unable to move to allow the lysine in to the allosteric site due to the slight variation in Y107, as these two residues are connected via hydrophobic stacking. Overall when the water channel was blocked the catalysis was slowed supporting the hypothesis that the water channel is involved in the allosteric mechanism of dihydrodipicolinate synthase.