The importance of the link between Glu204 of the catalytic chain and Arg130 of the regulatory chain for the homotropic and heterotropic properties of Escherichia coli aspartate transcarbamoylase.
Recent x-ray crystallographic studies of aspartate transcarbamoylase bound with CTP have detected molecular asymmetry in the interface between the catalytic and regulatory subunits (Kim, K. H., Pan, Z., Honzatko, R. B., Ke, H.-M., and Lipscomb, W. N. (1987) J. Mol. Biol. 196, 863-875). In three of the six interfaces, a salt link occurs between Arg130 of the regulatory chain and Glu204 of the catalytic chain; however, these same residues are 15 A apart in the other three interfaces. In order to determine if this is important for the function of the enzyme, two mutant versions of aspartate transcarbamoylase were created by site-specific mutagenesis. Glu204 of the catalytic chain was converted to a glutamine (Glu204c----Gln) and Arg130 of the regulatory chain was converted to a glycine (Arg130r----Gly). The thermal stability of the Arg130r----Gly enzyme is dramatically reduced, whereas the thermal stability of the Glu204c----Gln enzyme is unaltered compared to the wild-type enzyme. The maximal velocity of both mutant enzymes is identical with that of the wild-type enzyme, however both mutant enzymes have altered substrate affinity and regulatory properties. Based on these studies, the link between Glu204 of the catalytic chain and Arg130 of the regulatory chain is important for the heterotropic properties of the enzyme. Furthermore, the interface between the domain of the regulatory chain which binds zinc and the domain of the catalytic chain which binds aspartate may be more important for CTP inhibition than ATP activation. These data also suggest that heterotropic cooperativity is very sensitive to alterations in the catalytic-regulatory interface. However, no clear relationship has been observed between the structural asymmetry and the function of the enzyme.
Stebbins
J W
JW
Department of Chemistry, Boston College, Massachusetts 02167.
Kantrowitz
E R
ER
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D001221:Aspartate Carbamoyltransferase / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D014961:X-Ray Diffraction
D005971:Glutamates; D005973:Glutamine; D018698:Glutamic Acid; D001120:Arginine; D001221:Aspartate Carbamoyltransferase