Crystal structures of phosphonoacetamide ligated T and phosphonoacetamide and malonate ligated R states of aspartate carbamoyltransferase at 2.8-A resolution and neutral pH.
The T----R transition of the cooperative enzyme aspartate carbamoyltransferase occurs at pH 7 in single crystals without visibly cracking many of the crystals and leaving those uncracked suitable for single-crystal X-ray analysis. To promote the T----R transition, we employ the competitive inhibitors of carbamoyl phosphate and aspartate, which are phosphonoacetamide (PAM) and malonate, respectively. In response to PAM binding to the T-state crystals, residues Thr 53-Thr 55 and Pro 266-Pro 268 move to their R-state positions to bind to the phosphonate and amino group of PAM. These changes induce a conformation that can bind tightly the aspartate analogue malonate, which thereby effects the allosteric transition. We prove this by showing that PAM-ligated T-state crystals (Tpam), space group P321 (a = 122.2 A, c = 142.2 A), when transferred to a solution containing 20 mM PAM and 8 mM malonate at pH 7, isomerize to R-state crystals (Rpam,mal,soak), space group also P321 (a = 122.2 A, c = 156.4 A). The R-state structure in which the T----R transition occurs within the crystal at pH 7 compares very well (rms = 0.19 A for all atoms) with an R-state structure determined at pH 7 in which the crystals were initially grown in a solution of PAM and malonate at pH 5.9 and subsequently transferred to a buffer containing the ligands at pH 7 (Rpam,mal,crys). In fact, both of the PAM and malonate ligated R-state structures are very similar to both the carbamoyl phosphate and succinate or the N-(phosphonoacetyl)-L-aspartate ligated structures, even though the R-state structures reported here were determined at pH 7. Crystallographic residuals refined to 0.16-0.18 at 2.8-A resolution for the three structures.
Gouaux
J E
JE
Gibbs Chemical Laboratory, Harvard University, Cambridge, Massachusetts 02138.
Lipscomb
W N
WN
D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003201:Computers; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008314:Malonates / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009943:Organophosphorus Compounds / Q000378:metabolism*; D011487:Protein Conformation; D014961:X-Ray Diffraction
D008314:Malonates; D009943:Organophosphorus Compounds; C063084:phosphonoacetamide; C030290:malonic acid; D001221:Aspartate Carbamoyltransferase