The conserved residues glutamate-37, aspartate-100, and arginine-269 are important for the structural stabilization of Escherichia coli aspartate transcarbamoylase.
Aspartate transcarbamoylase from Escherichia coli is a dodecameric enzyme consisting of two trimeric catalytic subunits and three dimeric regulatory subunits. The X-ray structure of this enzyme indicates that the side chains of His-41, Asp-100, and Asp-90 from one catalytic chain form interactions with the side chains of Glu-37, Arg-65, and Arg-269, respectively, from an adjacent catalytic chain. In order to determine whether these interactions are important for the structural stabilization of the enzyme and/or homotropic and heterotropic effects, four mutant versions of aspartate transcarbamoylase, Glu-37-->Ala, Asp-100-->Asn, Asp-100-->Ala, and Arg-269-->Ala, were created by site-specific mutagenesis. The Glu-37-->Ala holoenzyme exhibits essentially wild-type behavior with respect to homotropic cooperativity and heterotropic regulation by ATP and CTP. The Glu-37-->Ala catalytic subunit exhibits a half-life of inactivation at 69 +/- 0.5 degrees C of 4.9 min, as compared to 5.8 min for the wild-type catalytic subunit. The Asp-100-->Asn and Asp-100-->Ala holoenzymes are slightly more active than the wild-type holoenzyme, exhibit 1.4-fold and 1.8-fold reductions in the aspartate concentration at half the maximal specific activity, respectively, and show increased affinities for ATP and CTP. Both the Asp-100-->Asn and Asp-100--> Ala catalytic subunits exhibit a 2-fold reduction in the half-life of inactivation at 69 +/- 0.5 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Baker
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Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167.
Kantrowitz
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D000595:Amino Acid Sequence; D001120:Arginine; D001221:Aspartate Carbamoyltransferase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid; D017124:Conserved Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism
D005971:Glutamates; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D001120:Arginine; D001221:Aspartate Carbamoyltransferase