1ggo
From Proteopedia
(New page: 200px<br /><applet load="1ggo" size="450" color="white" frame="true" align="right" spinBox="true" caption="1ggo, resolution 2.60Å" /> '''T453A MUTANT OF PYRU...) |
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| - | [[Image:1ggo.gif|left|200px]]<br /><applet load="1ggo" size=" | + | [[Image:1ggo.gif|left|200px]]<br /><applet load="1ggo" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1ggo, resolution 2.60Å" /> | caption="1ggo, resolution 2.60Å" /> | ||
'''T453A MUTANT OF PYRUVATE, PHOSPHATE DIKINASE'''<br /> | '''T453A MUTANT OF PYRUVATE, PHOSPHATE DIKINASE'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Potential domain-domain docking residues, identified from the x-ray | + | Potential domain-domain docking residues, identified from the x-ray structure of the Clostridium symbiosum apoPPDK, were replaced by site-directed mutagenesis. The steady-state and transient kinetic properties of the mutant enzymes were determined as a way of evaluating docking efficiency. PPDK mutants, in which one of two stringently conserved docking residues located on the N-terminal domain (Arg(219) and Glu(271)) was substituted, displayed largely unimpeded catalysis of the phosphoenolpyruvate partial reaction at the C-terminal domain, but significantly impaired catalysis (>10(4)) of the ATP pyrophosphorylation of His(455) at the N-terminal domain. In contrast, alanine mutants of two potential docking residues located on the N-terminal domain (Ser(262) and Lys(149)), which are not conserved among the PPDKs, exhibited essentially normal catalytic turnover. Arg(219) and Glu(271) were thus proposed to play an important role in guiding the central domain and, hence, the catalytic His(455) into position for catalysis. Substitution of central domain residues Glu(434)/Glu(437) and Thr(453), the respective docking partners of Arg(219) and Glu(271), resulted in mutants impaired in catalysis at the ATP active site. The x-ray crystal structure of the apo-T453A PPDK mutant was determined to test for possible misalignment of residues at the N-terminal domain-central domain interface that might result from loss of the Thr(453)-Glu(271) binding interaction. With the exception of the mutation site, the structure of T453A PPDK was found to be identical to that of the wild-type enzyme. It is hypothesized that the two Glu(271) interfacial binding sites that remain in the T453A PPDK mutant, Thr(453) backbone NH and Met(452) backbone NH, are sufficient to stabilize the native conformation as observed in the crystalline state but may be less effective in populating the reactive conformation in solution. |
==About this Structure== | ==About this Structure== | ||
| - | 1GGO is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Clostridium_symbiosum Clostridium symbiosum] with SO4 as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Pyruvate,_phosphate_dikinase Pyruvate, phosphate dikinase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.9.1 2.7.9.1] Full crystallographic information is available from [http:// | + | 1GGO is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Clostridium_symbiosum Clostridium symbiosum] with <scene name='pdbligand=SO4:'>SO4</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Pyruvate,_phosphate_dikinase Pyruvate, phosphate dikinase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.9.1 2.7.9.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1GGO OCA]. |
==Reference== | ==Reference== | ||
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[[Category: transferase]] | [[Category: transferase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:49:51 2008'' |
Revision as of 10:49, 21 February 2008
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T453A MUTANT OF PYRUVATE, PHOSPHATE DIKINASE
Overview
Potential domain-domain docking residues, identified from the x-ray structure of the Clostridium symbiosum apoPPDK, were replaced by site-directed mutagenesis. The steady-state and transient kinetic properties of the mutant enzymes were determined as a way of evaluating docking efficiency. PPDK mutants, in which one of two stringently conserved docking residues located on the N-terminal domain (Arg(219) and Glu(271)) was substituted, displayed largely unimpeded catalysis of the phosphoenolpyruvate partial reaction at the C-terminal domain, but significantly impaired catalysis (>10(4)) of the ATP pyrophosphorylation of His(455) at the N-terminal domain. In contrast, alanine mutants of two potential docking residues located on the N-terminal domain (Ser(262) and Lys(149)), which are not conserved among the PPDKs, exhibited essentially normal catalytic turnover. Arg(219) and Glu(271) were thus proposed to play an important role in guiding the central domain and, hence, the catalytic His(455) into position for catalysis. Substitution of central domain residues Glu(434)/Glu(437) and Thr(453), the respective docking partners of Arg(219) and Glu(271), resulted in mutants impaired in catalysis at the ATP active site. The x-ray crystal structure of the apo-T453A PPDK mutant was determined to test for possible misalignment of residues at the N-terminal domain-central domain interface that might result from loss of the Thr(453)-Glu(271) binding interaction. With the exception of the mutation site, the structure of T453A PPDK was found to be identical to that of the wild-type enzyme. It is hypothesized that the two Glu(271) interfacial binding sites that remain in the T453A PPDK mutant, Thr(453) backbone NH and Met(452) backbone NH, are sufficient to stabilize the native conformation as observed in the crystalline state but may be less effective in populating the reactive conformation in solution.
About this Structure
1GGO is a Single protein structure of sequence from Clostridium symbiosum with as ligand. Active as Pyruvate, phosphate dikinase, with EC number 2.7.9.1 Full crystallographic information is available from OCA.
Reference
Identification of domain-domain docking sites within Clostridium symbiosum pyruvate phosphate dikinase by amino acid replacement., Wei M, Li Z, Ye D, Herzberg O, Dunaway-Mariano D, J Biol Chem. 2000 Dec 29;275(52):41156-65. PMID:10995759
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