1tum
From Proteopedia
(New page: 200px<br /><applet load="1tum" size="450" color="white" frame="true" align="right" spinBox="true" caption="1tum" /> '''MUTT PYROPHOSPHOHYDROLASE-METAL-NUCLEOTIDE-M...) |
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'''MUTT PYROPHOSPHOHYDROLASE-METAL-NUCLEOTIDE-METAL COMPLEX, NMR, 16 STRUCTURES'''<br /> | '''MUTT PYROPHOSPHOHYDROLASE-METAL-NUCLEOTIDE-METAL COMPLEX, NMR, 16 STRUCTURES'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The MutT enzyme (129 residues) catalyzes the hydrolysis of nucleoside | + | The MutT enzyme (129 residues) catalyzes the hydrolysis of nucleoside triphosphates (NTP) by substitution at the rarely attacked beta-P, to yield NMP and pyrophosphate. It requires two divalent cations, forming an active E-M2+-NTP-M2+ complex. The solution structure of the free enzyme consists of a five-stranded mixed beta-sheet connected by loop I-alpha-helix I-loop II, by two tight turns, and by loop III and terminated by loop IV-alpha-helix II [Abeygunawardana, C., et al. (1995) Biochemistry 34, 14997-15005]. Assignments of backbone 15N and NH resonances and side chain 15N and NH2 resonances of the quaternary complex were made by 1H-15N HSQC titrations of the free enzyme with MgCl2 followed by equimolar AMPCPP/MgCl2. H(alpha) assignments were made by 1H-15N 3D TOCSY HSQC, and 1H-13C CT-HSQC spectra and backbone and side chain 1H and 13C assignments were made by 3D HCCH TOCSY experiments. Ligands donated by the protein to the enzyme-bound divalent cation, identified by paramagnetic effects of Co2+ and Mn2+ on CO(C)H spectra, are the carboxylate groups of Glu-56, -57, and -98 and the amide carbonyl of Gly-38. The solution structure of the complex was computed with XPLOR using a total of 2168 NOE and 83 phi restraints for the protein, 11 intramolecular NOEs for bound Mg2+ AMPCPP, 22 intermolecular NOEs between MutT and AMPCPP, and distances from the enzyme-bound Co2+ to the three phosphorus atoms of Co3+(NH3)4AMPCPP from paramagnetic effects of Co2+ on their T1 values. The fold of the MutT enzyme in the complex is very similar to that of the free enzyme, with minor changes in the metal and substrate binding sites. The adenine ring binds in a hydrophobic cleft, interacting with Leu-4 and Ile-6 on beta-strand A and with Ile-80 on beta-strand D. The 6-NH2 group of adenine approaches the side chain NH2 of Asn-119. This unfavorable interaction is consistent with the stronger binding by MutT of guanine nucleotides, which have a 6-keto group. The ribose binds with its hydroxyl groups oriented toward the solvent and its hydrophobic face interacting with Leu-4, Ile-6, and the gamma-CH2 of Lys-39 of loop I. The metal-triphosphate moiety appears to bind in the second coordination sphere of the enzyme-bound divalent cation. One of two intervening water ligands is well positioned to attack P(beta) with inversion and to donate a hydrogen bond to the conserved residue, Glu-53, which may deprotonate or orient the attacking water ligand. Lys-39 which is positioned to interact electrostatically with the alpha-phosphoryl group may facilitate the departure of the leaving NMP. On the basis of the structure of the quaternary complex, a mechanism of the MutT reaction is proposed which is qualitatively and quantitatively consistent with kinetic and mutagenesis studies. It is suggested that similar mechanisms may be operative for other enzymes that catalyze substitution at P(beta) of NTP substrates. |
==About this Structure== | ==About this Structure== | ||
| - | 1TUM is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with MG, APC and CON as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http:// | + | 1TUM is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=MG:'>MG</scene>, <scene name='pdbligand=APC:'>APC</scene> and <scene name='pdbligand=CON:'>CON</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1TUM OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Abeygunawardana, C.]] | [[Category: Abeygunawardana, C.]] | ||
| - | [[Category: Bessman, M | + | [[Category: Bessman, M J.]] |
| - | [[Category: Frick, D | + | [[Category: Frick, D N.]] |
[[Category: Lin, J.]] | [[Category: Lin, J.]] | ||
| - | [[Category: Mildvan, A | + | [[Category: Mildvan, A S.]] |
[[Category: APC]] | [[Category: APC]] | ||
[[Category: CON]] | [[Category: CON]] | ||
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[[Category: quaternary complex]] | [[Category: quaternary complex]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 15:17:30 2008'' |
Revision as of 13:17, 21 February 2008
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MUTT PYROPHOSPHOHYDROLASE-METAL-NUCLEOTIDE-METAL COMPLEX, NMR, 16 STRUCTURES
Overview
The MutT enzyme (129 residues) catalyzes the hydrolysis of nucleoside triphosphates (NTP) by substitution at the rarely attacked beta-P, to yield NMP and pyrophosphate. It requires two divalent cations, forming an active E-M2+-NTP-M2+ complex. The solution structure of the free enzyme consists of a five-stranded mixed beta-sheet connected by loop I-alpha-helix I-loop II, by two tight turns, and by loop III and terminated by loop IV-alpha-helix II [Abeygunawardana, C., et al. (1995) Biochemistry 34, 14997-15005]. Assignments of backbone 15N and NH resonances and side chain 15N and NH2 resonances of the quaternary complex were made by 1H-15N HSQC titrations of the free enzyme with MgCl2 followed by equimolar AMPCPP/MgCl2. H(alpha) assignments were made by 1H-15N 3D TOCSY HSQC, and 1H-13C CT-HSQC spectra and backbone and side chain 1H and 13C assignments were made by 3D HCCH TOCSY experiments. Ligands donated by the protein to the enzyme-bound divalent cation, identified by paramagnetic effects of Co2+ and Mn2+ on CO(C)H spectra, are the carboxylate groups of Glu-56, -57, and -98 and the amide carbonyl of Gly-38. The solution structure of the complex was computed with XPLOR using a total of 2168 NOE and 83 phi restraints for the protein, 11 intramolecular NOEs for bound Mg2+ AMPCPP, 22 intermolecular NOEs between MutT and AMPCPP, and distances from the enzyme-bound Co2+ to the three phosphorus atoms of Co3+(NH3)4AMPCPP from paramagnetic effects of Co2+ on their T1 values. The fold of the MutT enzyme in the complex is very similar to that of the free enzyme, with minor changes in the metal and substrate binding sites. The adenine ring binds in a hydrophobic cleft, interacting with Leu-4 and Ile-6 on beta-strand A and with Ile-80 on beta-strand D. The 6-NH2 group of adenine approaches the side chain NH2 of Asn-119. This unfavorable interaction is consistent with the stronger binding by MutT of guanine nucleotides, which have a 6-keto group. The ribose binds with its hydroxyl groups oriented toward the solvent and its hydrophobic face interacting with Leu-4, Ile-6, and the gamma-CH2 of Lys-39 of loop I. The metal-triphosphate moiety appears to bind in the second coordination sphere of the enzyme-bound divalent cation. One of two intervening water ligands is well positioned to attack P(beta) with inversion and to donate a hydrogen bond to the conserved residue, Glu-53, which may deprotonate or orient the attacking water ligand. Lys-39 which is positioned to interact electrostatically with the alpha-phosphoryl group may facilitate the departure of the leaving NMP. On the basis of the structure of the quaternary complex, a mechanism of the MutT reaction is proposed which is qualitatively and quantitatively consistent with kinetic and mutagenesis studies. It is suggested that similar mechanisms may be operative for other enzymes that catalyze substitution at P(beta) of NTP substrates.
About this Structure
1TUM is a Single protein structure of sequence from Escherichia coli with , and as ligands. Full crystallographic information is available from OCA.
Reference
Solution structure of the quaternary MutT-M2+-AMPCPP-M2+ complex and mechanism of its pyrophosphohydrolase action., Lin J, Abeygunawardana C, Frick DN, Bessman MJ, Mildvan AS, Biochemistry. 1997 Feb 11;36(6):1199-211. PMID:9063868
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