title: Function of serine-171 in domain closure, cooperativity, and catalysis in Escherichia coli aspartate transcarbamoylase.
abstract: Structural studies of Escherichia coli aspartate transcarbamoylase suggest that the R state of the enzyme is stabilized by an interaction between Ser-171 of the aspartate domain and both the backbone carbonyl of His-134 and the side chain of Gln-133 of the carbamoyl phosphate domain of a catalytic chain [Ke, H.-M., Lipscomb, W.N., Cho, Y., & Honzatko, R. B. (1988) J. Mol. Biol. 204, 725-747]. In the present study, site-specific mutagenesis is used to replace Ser-171 by alanine, thereby eliminating the interactions between Ser-171 and both Gln-133 and His-134. The Ser-171----Ala holoenzyme exhibits no cooperativity, more than a 140-fold loss of activity, little change in the carbamoyl phosphate concentration at half the maximal observed specific activity, and a 7-fold increase in the aspartate concentration at half the maximal observed specific activity. Although the Ser-171----Ala enzyme exhibits no homotropic cooperativity, it is still activated by N-(phosphonacetyl)-L-aspartate (PALA), but not by succinate, in the presence of saturating carbamoyl phosphate and subsaturating aspartate. At subsaturating concentrations of aspartate, the Ser-171----Ala enzyme is still activated by ATP but is inhibited less by CTP than is the wild-type enzyme. At saturating concentrations of aspartate, the Ser-171----Ala enzyme is activated by ATP and inhibited by CTP to an even greater extent than at subsaturating concentrations of aspartate. At saturating aspartate, the wild-type enzyme is neither activated by ATP nor inhibited by CTP.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02167.
authors.forename: N J
authors.initials: NJ
authors.lastname: Dembowski
authors.forename: C J
authors.initials: CJ
authors.lastname: Newton
authors.forename: E R
authors.initials: ER
authors.lastname: Kantrowitz
mesh_terms: D001221:Aspartate Carbamoyltransferase / Q000235:genetics* / Q000378:metabolism; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D009154:Mutation; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D011487:Protein Conformation; D012694:Serine; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D001224:Aspartic Acid; D012694:Serine; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid