THE STRUCTURAL BASIS FOR PSEUDOREVERSION OF THE H95N LESION BY THE SECONDARY S96P MUTATION IN TRIOSEPHOSPHATE ISOMERASE

The structural basis for the 3000-fold decrease in catalytic efficienc y of the H95N mutant chicken triosephosphate isomerase and the 60-fold regain of catalytic efficiency in the double mutant, H95N . S96P, hav e been analyzed. The results from a combination of X-ray crystallograp hy and Fourier transform infrared spectroscopy experiments indicate th at the predominant defect in the H95N mutant isomerase appears to be i ts inability to bind the substrate in a coplanar, cis conformation. Th e structures of each mutant isomerase were determined from X-ray cryst allography of the complex of phosphoglycolohydroxamate (PGH), an inter mediate analog, with the isomerase, and each was solved to a resolutio n of 1.9 Angstrom. The PGH appeared to be in two different conformatio ns in which the enediol-mimicking atoms, O2-N2-C1-O1, of the PGH were not coplanar. No density was observed that would correspond to the cop lanar conformation. Two bands are observed for the dihydroxyacetone ph osphate carbonyl in the H95N mutant FTIR spectrum, and these can be ex plained if the O1 of DHAP, like the O1 of PGH in the crystal structure , is in two different positions. Two ordered water molecules are locat ed between O1 of PGH and N delta of N95. Comparison of the structure o f the pseudorevertant, H95N . S96P with that for the H95N single mutan t, shows that S96P mutation causes the double mutant to regain the abi lity to bind PGH predominantly in the coplanar, cis conformation. Elec tron density for a single ordered water molecule bridging the N95 amid e side chain and the O2 of PGH is observed, but the density was weak, perhaps indicating that the water molecule is somewhat disordered. Whe ther or not a water molecule is hydrogen bonded to O2 of PGH may expla in the two carbonyl stretching frequencies observed for the GAP carbon yl. Together, the crystal structures and the FTIR data allow a complet e explanation of the catalytic properties of these two mutant isomeras es.