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Weakening of the interface between adjacent catalytic chains promotes domain closure in escherichia coli aspartate transcarbamoylase
Author(s) -
Baker Darren P.,
Keiser Richard T.,
Kantrowitz Evan R.,
Fetler Luc,
Vachette Patrice
Publication year - 1995
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560040212
Subject(s) - aspartate carbamoyltransferase , cooperativity , enzyme , biochemistry , escherichia coli , mutant , chemistry , stereochemistry , allosteric regulation , gene
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 52 :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.