TY - JOUR
T1 - Crystal Structures of Phosphonoacetamide Ligated T and Phosphonoacetamide and Malonate Ligated R States of Aspartate Carbamoyltransferase at 2.8-Å Resolution and Neutral pH
AU - Eric Gouaux, J.
AU - Lipscomb, William N.
PY - 1990/1/1
Y1 - 1990/1/1
N2 - 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 Å, c = 142.2 Å), 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 Å, c = 156.4 Å). The R-state structure in which the T → R transition occurs within the crystal at pH 7 compares very well (rms = 0.19 Å 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-Å resolution for the three structures.
AB - 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 Å, c = 142.2 Å), 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 Å, c = 156.4 Å). The R-state structure in which the T → R transition occurs within the crystal at pH 7 compares very well (rms = 0.19 Å 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-Å resolution for the three structures.
UR - http://www.scopus.com/inward/record.url?scp=0025016827&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0025016827&partnerID=8YFLogxK
U2 - 10.1021/bi00454a013
DO - 10.1021/bi00454a013
M3 - Article
C2 - 2405902
AN - SCOPUS:0025016827
SN - 0006-2960
VL - 29
SP - 389
EP - 402
JO - Biochemistry
JF - Biochemistry
IS - 2
ER -