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Site-directed alterations to the geometry of the aspartate transcarbamoylase zinc domain: Selective alteration to regulation by heterotropic ligands, isoelectric point, and stability in urea




, : Site-directed alterations to the geometry of the aspartate transcarbamoylase zinc domain: Selective alteration to regulation by heterotropic ligands, isoelectric point, and stability in urea. Biochemistry 32(16): 4156-4167

Structural aspects requisite for allosteric function in the regulatory chain of aspartate transcarbamoylase were explored by site-specific amino acid insertion or substitution within the zinc domain in the region of contact between the catalytic and regulatory chains. Amino acid substitution at two positions yielded enzymes which retained a maximum velocity similar to that of the wild-type enzyme but responded differently from the native enzyme in the presence of regulatory nucleoside triphosphates. A change of zinc coordinate amino acid C109 to histidine and a change of El19 to aspartic acid resulted in enzymes which demonstrated synergistic inhibition by CTP and UTP but not inhibition by CTP in either phosphate buffer or a morpholino-based tripartate (TP) buffer at pH 7. At pH 8.3, where there is a higher proportion of T-state conformers in the native enzyme, the mutants diverged from their similar kinetic behavior. C109H remained an enzyme which was not inhibited by CTP but was still inhibited by CTP+UTP. E119D was inhibited by both CTP and CTP+UTP. Activation of the mutants by ATP was found to vary either with pH or with phosphate as a buffer component. C109H was activated by ATP in phosphate, while in TP at either pH 7 or 8.3 its activation by ATP was diminished or absent. E119D was activated by ATP in phosphate at pH 7 or in TP at pH 8.3, but not in TP at pH 7. In TP at pH 7, where neither mutant was activated by ATP, the S-0.5 values and Hill coefficients of the unliganded mutant enzymes resembled those of the ATP-liganded wild-type enzyme. While neither mutation would be predicted to alter the net charge of the holoenzyme, differences in the isolectric point of the mutants were observed if phosphate was present. This result suggests that the isoelectric point of aspartate transcarbamoylase is conformationally dependent and that the mutants exist in an altered conformation. In addition, the stabilities of both mutant holoenzymes were reduced substantially from those of the wild-type enzyme in 4 M urea. C109H was more stable at pH 8.25 in a Tris buffer; E119D was more stable at pH 7 in the phosphate buffer. Potential effects of these mutations on the active site chemistry and geometry are discussed.

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