Abstract
Gln34, Gln224, Leu228, and Ser240 are conserved residues in the vicinity of bound IMP in the crystal structure of Escherichia coli adenylosuccinate synthetase. Directed mutations were carried out, and wild-type and mutant enzymes were purified to homogeneity. Circular dichroism spectroscopy indicated no difference in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. Mutants L228A and S240A exhibited modest changes in their initial rate kinetics relative to the wild-type enzyme, suggesting that neither Leu228 nor Ser240 play essential roles in substrate binding or catalysis. The mutants Q224M and Q224E exhibited no significant change in KmGTP and KmASP and modest changes in KmIMP relative to the wild-type enzyme. However, kcat decreased 13-fold for the Q224M mutant 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 emission fluorescence spectra of Q224M, Q224E, and wild-type proteins under denaturing conditions indicate comparable stabilities. Mutant Q34E exhibits a 60-fold decrease in kcat compared with that of the wild-type enzyme, which is attributed to the disruption of the Gln34 to Gln224 hydrogen bond observed in crystal structures. Presented here is a mechanism for the synthetase, whereby Gln224 works in concert with Asp13 to stabilize the 6-oxyanion of IMP.
Highlights
Known primary sequences of AMPSase, including those of bacteria and mammals [7,8,9,10,11,12,13,14,15] are 40 –90% identical, implying similar structure and function for this protein, regardless of source
The chemical mechanism proposed for the synthetase by Lieberman [21] and Fromm [22] calls for the formation of 6-phosphoryl-IMP [19, 23, 24] as an intermediate in the catalytic reaction
Residues located in the vicinity of C-6 of IMP should be important in catalysis and/or substrate binding
Summary
Materials—GTP, IMP, L-aspartate, adenylosuccinate, phenylmethylsulfonyl fluoride, and bovine serum albumin were obtained from Sigma. A site-directed mutagenesis kit was obtained from Amersham Corp. E. coli strain XL-1 blue was obtained from Stratagene. An E. coli purA2 strain H1238 (thr-25, tonA49, argF58, purA54, argI61) and an E. coli purB2 strain H680 (fhuA2, lacY1, tsx-70, glnV44(AS), gal-6, l-, purB51, trpC45, his-68, tyrA2, rspL125(strR), malT1(lR), xylA7, mtlA2, thi-1) were obtained from Dr B. 0.45-mm polyvinylidene difluoride membranes were obtained from Millipore Corp. Other reagents and chemicals were obtained from Sigma.
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