RELATIONSHIP OF CONSERVED RESIDUES IN THE IMP BINDING-SITE TO SUBSTRATE RECOGNITION AND CATALYSIS IN ESCHERICHIA-COLI ADENYLOSUCCINATE SYNTHETASE

Gln(34), Gln(224), Leu(228), and Ser(240) are conserved residues in th e vicinity of bound IMP in the crystal structure of Escherichia coli a denylosuccinate synthetase. Directed mutations were carried out, and w ild-type and mutant enzymes were purified to homogeneity. Circular dic hroism spectroscopy indicated no difference in secondary structure bet ween the mutants and the wildtype enzyme in the absence of substrates. Mutants L228A and S240A exhibited modest changes in their initial rat e kinetics relative to the wild-type enzyme, suggesting that neither L eu(228) nor Ser(240) play essential roles in substrate binding or cata lysis. The mutants Q224M and Q224E exhibited no significant change in K-m(GTP) and K-m(ASP) and modest changes in K-m(IMP) relative to the w ild-type enzyme. However, k(cat) decreased 13-fold for the Q224M mutan t and 10(4)-fold for the Q224E mutant relative to the wild-type enzyme . Furthermore, the Q224E mutant showed an optimum pH at 6.2, which is 1.5 pH units lower than that of the wild-type enzyme. Tryptophan emiss ion fluorescence spectra of Q224M, Q224E, and wild-type proteins under denaturing conditions indicate comparable stabilities. Mutant Q34E ex hibits a 60-fold decrease in k(cat) compared with that of the wild-typ e enzyme, which is attributed to the disruption of the Gln(34) to Gln( 224) hydrogen bond observed in crystal structures. Presented here is a mechanism for the synthetase, whereby Gln(224) works in concert with Asp(13) to stabilize the B-oxyanion of IMP.