2at1
CRYSTAL STRUCTURES OF PHOSPHONOACETAMIDE LIGATED T AND PHOSPHONOACETAMIDE AND MALONATE LIGATED R STATES OF ASPARTATE CARBAMOYLTRANSFERASE AT 2.8-ANGSTROMS RESOLUTION AND NEUTRAL PH
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OverviewOverview
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.
About this StructureAbout this Structure
2AT1 is a Protein complex structure of sequences from Escherichia coli with MAL, ZN and PCT as ligands. Active as Aspartate carbamoyltransferase, with EC number 2.1.3.2 Full crystallographic information is available from OCA.
ReferenceReference
Crystal structures of phosphonoacetamide ligated T and phosphonoacetamide and malonate ligated R states of aspartate carbamoyltransferase at 2.8-A resolution and neutral pH., Gouaux JE, Lipscomb WN, Biochemistry. 1990 Jan 16;29(2):389-402. PMID:2405902
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