FUNCTION OF SERINE-171 IN DOMAIN CLOSURE, COOPERATIVITY, AND CATALYSIS IN ESCHERICHIA-COLI ASPARTATE TRANSCARBAMOYLASE

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 Gin-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 Gin-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. The heterotropic effectors ATP and CTP alter the maximal velocity of the Ser-171 → Ala enzyme but do not affect the aspartate concentration at half the maximal observed specific activity. For the wild-type enzyme, the maximal velocity remains unaltered in the presence of either ATP or CTP, but these nucleotides do alter the aspartate concentration at half the maximal observed specific activity. The isolated catalytic subunit of the Ser-171 → Ala enzyme is only 6-fold less active than the wild-type catalytic subunit, and the mutant catalytic subunit is 32-fold more active than the mutant holoenzyme. The Km for aspartate is 180 mM for the Ser-171 → Ala catalytic subunit compared to 6.0 mM for the wild-type catalytic subunit. Analysis of these data suggests that the Ser-171 → Ala enzyme exists in a low-activity and low-affinity state even when saturated with substrates, implying that the interdomain bridging interactions between Ser-171 and both the backbone carbonyl of His-134 and the side chain of Gln-133 are important for the formation of the high-activity high-affinity state of the enzyme. Furthermore, these data support the proposal [Ladjimi, M. M., Middleton, S. A., Kelleher, K. S., & Kantrowitz, E. R. (1988) Biochemistry 27, 268–276] that the closure of the two domains of the catalytic chain of the enzyme is involved in the creation of the high-affinity aspartate binding site. © 1990, American Chemical Society. All rights reserved.