The structural basis for pseudoreversion of the E165D lesion by the secondary S96P mutation in triosephosphate isomerase depends on the positions of active site water molecules.

摘要: The structural basis for the improvement in catalytic efficiency of mutant E165D chicken triosephosphate isomerase by secondary mutation, S96P, has been analyzed using a combination X-ray crystallography and Fourier transform infrared spectroscopy. All structures were complex phosphoglycolohydroxamate (PGH), an intermediate analog, with isomerase, each was solved to resolution 1.9 A. Comparison structure double mutant, E165D.S96P, that single E165D, as well wild-type shows only insignificant differences positions side chains all mutants when compared except both E165D.S96P mutants, aspartate chain approximately 0.7 A further away from substrate analog than glutamate chain. Significant observed crystal ordered water molecules bound at active site. loss two located near position 165 isomerases containing S96P mutation. resulting increase hydrophobicity pocket probably causes pKa base, D165, thereby improving its basicity. new molecule underneath PGH structure, which likely decreases pKa's protons, increasing their acidity. An enzyme derived carbonyl stretch 1746 cm-1 is IR spectrum substrates assigned stable ground state protonated D165-enediol(ate) complex. Thus, gain activity second site change results basicity enzyme, acidity stabilization reaction intermediate. three these effects seem be caused changes molecules.