Function of serine-171 in domain closure, cooperativity, and catalysis in Escherichia coli aspartate transcarbamoylase

• Published in: Biochemistry (Easton) Vol. 29; no. 15; pp. 3716 - 3723
• Main Authors: Dembowski, Niki J, Newton, Colin J, Kantrowitz, Evan R
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 17.04.1990
• Subjects: Analytical, structural and metabolic biochemistry; aspartate carbamoyltransferase; Aspartate Carbamoyltransferase - genetics; Aspartate Carbamoyltransferase - metabolism; Aspartic Acid - analogs & derivatives; Aspartic Acid - metabolism; Aspartic Acid - pharmacology; Binding Sites; Biological and medical sciences; Catalysis; Enzymes and enzyme inhibitors; Escherichia coli - drug effects; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; Kinetics; Mutation; Phosphonoacetic Acid - analogs & derivatives; Phosphonoacetic Acid - pharmacology; Protein Conformation; Serine; Structure-Activity Relationship; Substrate Specificity; Transferases; Escherichia coli; Serine; Site specificity; Enzymatic activity; Structure activity relation; Aspartate carbamoyltransferase; Bacteria; Oligonucleotide; Mutation; Kinetic parameter; Enterobacteriaceae; Conformation; Crystalline structure
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00467a018

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.