Weakening of the interface between adjacent catalytic chains promotes domain closure in escherichia coli aspartate transcarbamoylase

Abstract

Aspartate transcarbamoylase from Escherichia coli is a dodecameric enzyme consisting of two trimeric catalytic subunits and three dimeric regulatory subunits. Asp-100, from one catalytic chain, is involved in stabilizing the C1-C2 interface by means of its interaction with Arg-65 from an adjacent catalytic chain. Replacement of Asp-100 by Ala has been shown previously to result in increases in the maximal specific activity, homotropic cooperativity, and the affinity for aspartate (Baker DP, Kantrowitz ER, 1993, Biochemistry 32:10150-10158). In order to determine whether these properties were due to promotion of domain closure induced by the weakening of the C1-C2 interface, we constructed a double mutant version of aspartate transcarbamoylase in which the Asp-100 + Ala mutation was introduced into the Glu-50 -+ Ala holoenzyme, a mutant in which domain closure is impaired.The Glu-5O/Asp-100 + Ala enzyme is fourfold more active than the Glu-50 -+ Ala enzyme, and exhibits significant restoration of homotropic cooperativity with respect to aspartate. In addition, the Asp-IO0 -+ Ala mutation restores the ability of the Glu-50 -+ Ala enzyme to be activated by succinate and increases the affinity of the enzyme for the bisubstrate analogue N-(phosphonacety1)-L-aspartate (PALA). At subsaturating concentrations of aspartate, the Glu-50/Asp-100 -+ Ala enzyme is activated more by ATP than the Glu-50 + Ala enzyme and is also inhibited more by CTP than either the wild-type or the Glu-50 + Ala enzyme. As opposed to the wild-type enzyme, the Glu-50/Asp-100 + Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Glu-50/Asp-100 + Ala enzyme by solution X-ray scattering indicates that the double mutant exists in the same T quaternary structure as the wild-type enzyme in the absence of ligands and in the same R quaternary structure in the presence of saturating PALA. However, saturating concentrations of carbamoyl phosphate and succinate only convert a fraction of the Glu-50/Asp-100 -+ Ala enzyme population to the R quaternary structure, a behavior intermediate between that observed for the Glu-50 + Ala and wild-type enzymes. Solution X-ray scattering was also used to investigate the structural consequences of nucleotide binding to the Glu-50/Asp-100 + Ala enzyme.Keywords: domain closure; homotropic cooperativity; protein structure-function; site-specific mutagenesis; solution X-ray scattering *We dedicate this paper to the memory of our friend and colleague Escherichia coli aspartate transcarbamoylase (ATCase; EC Frederick C. Wedler, whose work on the catalytic mechanism of aspar-2.1.3.2) catalyzes the committed step in the biosynthesis of pytate transcarbamoylase has been fundamental to the understanding of rimidine nucleotides: the reaction between carbamoyl phosphate this complex enzyme. His personal and intellectual contributions to the scientific community will be sorely missed.and L-aspartate to form N-carbamoyl-L-aspartate and inorganic Reprint requ...

Use of L‐asparagine and N‐phosphonacetyl‐L‐asparagine to investigate the linkage of catalysis and homotropic cooperativity in E. coli aspartate transcarbomoylase

“…In the R‐state, Glu50 forms interdomain bridging interactions with Arg167 and Arg234, of the ASP domain, stabilizing the R‐state. Mutation of Glu50 to Ala results in a R‐state destabilized mutant with a 19‐fold reduction in activity compared to wild‐type 29. SAXS studies showed that upon addition of CP and succinate (an aspartate analog) the E50A mutant is in the T state in contrast to the wild‐type enzyme, which exists in the R state 29, 30.…”

“…Mutation of Glu50 to Ala results in a R‐state destabilized mutant with a 19‐fold reduction in activity compared to wild‐type 29. SAXS studies showed that upon addition of CP and succinate (an aspartate analog) the E50A mutant is in the T state in contrast to the wild‐type enzyme, which exists in the R state 29, 30. However, the E50A enzyme–PALA complex exists in the R structure similar to the structure of the wild‐type enzyme in the presence of PALA or substrates.…”

Characterization and assembly of the Pseudomonas aeruginosa aspartate transcarbamoylase-pseudo dihydroorotase complex

“…The three very important interactions between E. coli ATCase monomers in the catalytic trimer have been identified to be Lys-41, Asp-100, and Asp-90 of one monomer that form salt links with Glu-37, Arg-65, and Arg-269 of the adjacent subunit [36,37]. Multiple sequence alignment of P. aeruginosa ATCase with other trimeric ATCases (S1 Fig) showed that two of the three interactions are partially conserved in P. aeruginosa.…”