Triosephosphate Isomerase: Difference between revisions

The reaction mechanism for the isomerization of DHAP and G3P involves acid base catalysis proceeding through an enediol intermediate [ [http://en.wikipedia.org/wiki/Triose_phosphate_isomerase] ].  The active site residues, Glu165 and His95, were shown by early crystallization studies containing bound substrate or inhibitor, to be precisely positioned to serve as catalysts of the reaction. Glu165 initiates the first step of the reaction by abstracting a proton from the pro(R) position of carbon 1 of DHAP (rate determining step) as the oxygen of carbon 2 abstracts a proton from His95. This promotes rearrangement to the enediol intermediate. In the second step, His95 abstracts a proton from the oxygen of carbon 1 as the oxygen of carbon 2 abstracts a proton from Glu165. This step promotes rearrangement to the product, G3P, thereby regenerating the enzyme. The importance of these residues in catalysis was confirmed by the kinetic analysis of site-directed mutations. For example, catalytic activity decreases by orders of magnitude when Glu165 is mutated to Asn165. Additional studies using NMR spectroscopy have demonstrated a drastically lowered pKa for His95 and formation of a hydrogen bond with the substrate analogues’ carbonyl oxygen. Lys12 also plays an important role in catalysis. This residue is highly conserved, and electrostatic interactions with the bound substrate are implicated by x-ray crystallography. As a test of its functional role, Lys12 was mutated to methionine. The Met12 mutant enzyme was found to have a defect in substrate binding despite having a correctly folded active site and overall native protein structure. Steric hindrance was not an explanation for the defect in substrate binding, whereas the electrostatic environment of the active site was significantly altered for the Met12 mutant. Thus, Lys12 contributes an appropriate electrostatic environment in the active site to favor the binding of the negatively charged substrate.