title: Function of arginine-234 and aspartic acid-271 in domain closure, cooperativity, and catalysis in Escherichia coli aspartate transcarbamylase.
abstract: Two mutant versions of Escherichia coli aspartate transcarbamylase were created by site-specific mutagenesis. Arg-234 of the 240s loop was replaced by serine in order to help deduce the function of the interactions that normally occur between Arg-234 and both Glu-50 and Gln-231 in the R state of the enzyme. The other mutation involved the replacement of Asp-271 by asparagine to further test the functional importance of the Tyr-240-Asp-271 link that has previously been proposed to stabilize the T state of the enzyme [Middleton, S. A., & Kantrowitz, E. R. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5866-5870]. The Arg-234----Ser holoenzyme exhibits no cooperativity, a 24-fold reduction in maximal velocity, normal affinity for carbamyl phosphate, and substantially reduced affinity for aspartate and N-(phosphonoacetyl)-L-aspartate (PALA). Unlike the wild-type enzyme, the heterotropic effectors ATP and CTP are able to influence the activity of the Arg-234----Ser enzyme at saturating aspartate concentrations. The Arg-234----Ser catalytic subunit exhibits a 33-fold reduction in maximal activity, an aspartate Km of 261 mM, compared to 5.7 mM for the wild-type catalytic subunit, and only a small alteration in the Km for carbamyl phosphate. Together these results provide additional evidence that the interdomain bridging interactions between Glu-50 of the carbamyl phosphate domain and both Arg-167 and Arg-234 of the aspartate domain are necessary for the stabilization of the high-activity-high-affinity configuration of the active site of the enzyme. Furthermore, without the interdomain bridging interactions, the holoenzyme no longer exhibits homotropic cooperativity.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02167.
authors.forename: S A
authors.initials: SA
authors.lastname: Middleton
authors.forename: E R
authors.initials: ER
authors.lastname: Kantrowitz
mesh_terms: D001120:Arginine; D001216:Asparagine; D001221:Aspartate Carbamoyltransferase / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D012694:Serine
chemical_list: D001224:Aspartic Acid; D012694:Serine; D001216:Asparagine; D001120:Arginine; D001221:Aspartate Carbamoyltransferase