The role of water in the catalytic efficiency of triosephosphate isomerase

The structural basis for the effect of the S96P mutation in chicken triosep hosphate isomerase (cTIM) has been analyzed using a combination of X-ray cr ystallography and Fourier transform infrared spectroscopy. The X-ray struct ure is that of the enzyme complexed with phosphoglycolohydroxamate (PGH), a n intermediate analogue, solved at a resolution of 1.9 Angstrom. The S96P m utation was identified as a second-site reverent when catalytically cripple d mutants, E165D and H95N, were subjected to random mutagenesis. The presen ce of the second mutation leads to enhanced activity over the single mutati on. However, the effect of the S96P mutation alone is to decrease the catal ytic efficiency of the enzyme. The crystal structures of the S96P double mu tants show that this bulky proline side chain alters the water structure wi thin the active-site cavity (E165D; ref 1) and prevents nonproductive bindi ng conformations of the substrate (H95N; ref 2). Comparison of the S96P sin gle mutant structure with those of the wildtype cTIM, those of the single m utants (E165D and H95N), and those of the double mutants (E165D/ S96P and H 95N/S96P) begins to address the role of the conserved serine residue at thi s position. The results indicate that the residue positions the catalytic b ase E165 optimally for polarization of the substrate carbonyl, thereby aidi ng in proton abstraction. In addition, this residue is involved in position ing critical water molecules, thereby affecting the way in which water stru cture influences activity.