Crystal structures of phosphonoacetamide ligated T and phosphonoacetamide and malonate ligated R states of aspartate carbamoyltransferase at 2.8-Å resolution and neutral pH

Eric Gouaux, William N. Lipscomb

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71 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)389-402
Number of pages14
JournalBiochemistry
Volume29
Issue number2
StatePublished - 1990
Externally publishedYes

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Aspartate Carbamoyltransferase
Crystal structure
Crystals
Carbamyl Phosphate
NSC 224131
Aspartic Acid
Single crystals
Organophosphonates
X ray analysis
Succinic Acid
phosphonoacetamide
malonic acid
Conformations
Buffers
X-Rays
Ligands
Atoms

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{e8b7e672cc4147b28c5b1d4c98a1854e,
title = "Crystal structures of phosphonoacetamide ligated T and phosphonoacetamide and malonate ligated R states of aspartate carbamoyltransferase at 2.8-{\AA} resolution and neutral pH",
abstract = "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 {\AA}, c = 142.2 {\AA}), 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 {\AA}, c = 156.4 {\AA}). The R-state structure in which the T → R transition occurs within the crystal at pH 7 compares very well (rms = 0.19 {\AA} 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-{\AA} resolution for the three structures.",
author = "Eric Gouaux and Lipscomb, {William N.}",
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pages = "389--402",
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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 - Gouaux, Eric

AU - Lipscomb, William N.

PY - 1990

Y1 - 1990

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.

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