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

The structural basis for the improvement in catalytic efficiency of th e mutant E165D chicken triosephosphate isomerase by the secondary muta tion, S96P, has been analyzed using a combination of X-ray crystallogr aphy and Fourier transform infrared spectroscopy. All X-ray structures were of the complex of phosphoglycolohydroxamate (PGH), an intermedia te analog, with the isomerase, and each was solved to a resolution of 1.9 Angstrom. Comparison of the structure of the double mutant, E165D . S96P, with that of the single mutant, E165D, as well as with the wil d-type isomerase shows only insignificant differences in the positions of the side chains in all of the mutants when compared with the wild- type isomerase, except that in both the E165D and E165D . S96P mutants , the aspartate side chain was approximately 0.7 Angstrom further away from the substrate analog than the glutamate side chain, Significant differences were observed in the crystal structure of the E165D S96P d ouble mutant in the positions of ordered water molecules bound at the active site. The loss of two water molecules located near the side cha in at position 165 was observed in isomerases containing the S96P muta tion. The resulting increase in hydrophobicity of the pocket probably causes an increase in the pK(a) of the catalytic base, D165, thereby i mproving its basicity. A new ordered water molecule was observed under neath the bound PGH in the E165D . S96P structure, which likely decrea ses the pK(a)'s of the substrate protons, thereby increasing their aci dity. An enzyme derived carbonyl stretch at 1746 cm(-1) that is only o bserved in the IR spectrum of the E165D S96P double mutant isomerase w ith bound substrates has been assigned to a stable ground state proton ated D165-enediol(ate) intermediate complex. Thus, the gain in activit y resulting from the S96P second site change probably results from a c ombination of improving the basicity of the enzyme, improving the acid ity of the substrate protons, and stabilization of a reaction intermed iate. All three of these effects seem to be caused by changes in bound water molecules.