Monitoring the transition from the T to the R state in E.coli aspartate transcarbamoylase by X-ray crystallography: crystal structures of the E50A mutant enzyme in four distinct allosteric states.
A detailed description of the transition that allosteric enzymes undergo constitutes a major challenge in structural biology. We have succeeded in trapping four distinct allosteric states of a mutant enzyme of Escherichia coli aspartate transcarbomylase and determining their structures by X-ray crystallography. The mutant version of aspartate transcarbamoylase in which Glu50 in the catalytic chains was replaced by Ala destabilizes the native R state and shifts the equilibrium towards the T state. This behavior allowed the use of substrate analogs such as phosphonoacetamide and malonate to trap the enzyme in T-like and R-like structures that are distinct from the T-state structure of the wild-type enzyme (as represented by the structure of the enzyme with CTP bound and the R-state structure as represented by the structure with N-(phosphonacetyl)-L-aspartate bound). These structures shed light on the nature and the order of internal structural rearrangements during the transition from the T to the R state. They also suggest an explanation for diminished activity of the E50A enzyme and for the change in reaction mechanism from ordered to random for this mutant enzyme.
Stieglitz
Kimberly
K
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, MA 02467, USA.
Stec
Boguslaw
B
Baker
Darren P
DP
Kantrowitz
Evan R
ER
D000495:Allosteric Site; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry*; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology*; D008024:Ligands; D008314:Malonates / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009943:Organophosphorus Compounds / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012492:Salts / Q000737:chemistry; D013329:Structure-Activity Relationship; D014965:X-Rays
D008024:Ligands; D008314:Malonates; D009943:Organophosphorus Compounds; D012492:Salts; C063084:phosphonoacetamide; C030290:malonic acid; D001221:Aspartate Carbamoyltransferase