Weakening of the interface between adjacent catalytic chains promotes domain closure in Escherichia coli aspartate transcarbamoylase.
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-50/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-100-->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-(phosphonacetyl)-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.
Baker
D P
DP
Merkert Chemistry Center, Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02167-3860, USA.
Fetler
L
L
Keiser
R T
RT
Vachette
P
P
Kantrowitz
E R
ER
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000235:genetics*; D001224:Aspartic Acid / Q000378:metabolism; D002221:Carbamyl Phosphate / Q000378:metabolism; D003570:Cytidine Triphosphate / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D016296:Mutagenesis; D017354:Point Mutation; D011487:Protein Conformation; D012542:Scattering, Radiation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013386:Succinates / Q000494:pharmacology; D019802:Succinic Acid; D014544:Uridine Triphosphate / Q000494:pharmacology; D014965:X-Rays
D013386:Succinates; D001224:Aspartic Acid; D002221:Carbamyl Phosphate; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D019802:Succinic Acid; D001221:Aspartate Carbamoyltransferase; D014544:Uridine Triphosphate