The allosteric activator Mg-ATP modifies the quaternary structure of the R-state of Escherichia coli aspartate transcarbamylase without altering the T<-->R equilibrium.
The allosteric enzyme aspartate transcarbamylase from Escherichia coli (ATCase) displays regulatory properties that involve various conformational changes, including a large quaternary structure rearrangement. This entails a major change in its solution X-ray scattering curve upon binding substrate analogues. We show here that, in the presence of the nucleotide effector ATP, known to stimulate the enzyme activity, the scattering profiles show a marked dependence on the metal bound to ATP. Whereas ATP has no major effect on the scattering pattern of ATCase, a saturating concentration of Mg-ATP notably modifies the scattering profile of the enzyme, either in the absence or in the presence of the bisubstrate analogue N-(phosphonacetyl)-l-aspartate (PALA). The transition with PALA in the presence of this metal-nucleotide complex remains concerted. Furthermore, Mg-ATP, as already observed with ATP, has no detectable direct effect on the T to R transition. The experimental scattering curves in the presence of Mg-ATP were fitted by a modeling approach using rigid body movements of the regulatory subunits and the catalytic trimers in the crystal structures. While the differences observed in the T-state in the presence of Mg-ATP are essentially attributed to the binding per se of the nucleotide, the solution structure of the R-state complexed to Mg-ATP is even more extended along the 3-fold axis than the previously described R solution structure, which is already more stretched out along the same axis than the crystal R structure. Based on the crystal structure of the enzyme in the R-state complexed with free ATP, a proposal is made to account for the effect of magnesium.
Fetler
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Laboratoire de Biochimie des Signaux Régulateurs Cellulaires et Moléculaires, Université Pierre et Marie Curie, CNRS UMR 7631, 96, bd. Raspail, Paris, 75006, France.
Vachette
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D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D000494:Allosteric Regulation / Q000187:drug effects; D000495:Allosteric Site / Q000187:drug effects; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D002021:Buffers; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D020836:Protein Structure, Quaternary / Q000187:drug effects; D021122:Protein Subunits; D012996:Solutions; D013816:Thermodynamics; D014961:X-Ray Diffraction
D002021:Buffers; D020035:Holoenzymes; D008024:Ligands; D021122:Protein Subunits; D012996:Solutions; D001224:Aspartic Acid; C013195:sparfosic acid; D000255:Adenosine Triphosphate; D001221:Aspartate Carbamoyltransferase; D008274:Magnesium; D010746:Phosphonoacetic Acid