1c9v
From Proteopedia
(New page: 200px<br /><applet load="1c9v" size="450" color="white" frame="true" align="right" spinBox="true" caption="1c9v, resolution 1.70Å" /> '''H12A VARIANT OF RIBO...) |
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| - | [[Image:1c9v.gif|left|200px]]<br /><applet load="1c9v" size=" | + | [[Image:1c9v.gif|left|200px]]<br /><applet load="1c9v" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1c9v, resolution 1.70Å" /> | caption="1c9v, resolution 1.70Å" /> | ||
'''H12A VARIANT OF RIBONUCLEASE A'''<br /> | '''H12A VARIANT OF RIBONUCLEASE A'''<br /> | ||
==Overview== | ==Overview== | ||
| - | His12 and His119 are critical for catalysis of RNA cleavage by | + | His12 and His119 are critical for catalysis of RNA cleavage by ribonuclease A (RNase A). Substitution of either residue with an alanine decreases the value of k(cat)/K(M) by more than 10(4)-fold. His12 and His119 are proximal to the scissile phosphoryl group of an RNA substrate in enzyme-substrate complexes. Here, the role of these active site histidines in RNA binding was investigated by monitoring the effect of mutagenesis and pH on the stability of enzyme-nucleic acid complexes. X-ray diffraction analysis of the H12A and H119A variants at a resolution of 1.7 and 1.8 A, respectively, shows that the amino acid substitutions do not perturb the overall structure of the variants. Isothermal titration calorimetric studies on the complexation of wild-type RNase A and the variants with 3'-UMP at pH 6.0 show that His12 and His119 contribute 1.4 and 1.1 kcal/mol to complex stability, respectively. Determination of the stability of the complex of wild-type RNase A and 6-carboxyfluorescein approximately d(AUAA) at varying pHs by fluorescence anisotropy shows that the stability increases by 2.4 kcal/mol as the pH decreases from 8.0 to 4.0. At pH 4.0, replacing His12 with an alanine residue decreases the stability of the complex with 6-carboxyfluorescein approximately d(AUAA) by 2.3 kcal/mol. Together, these structural and thermodynamic data provide the first thorough analysis of the contribution of histidine residues to nucleic acid binding. |
==About this Structure== | ==About this Structure== | ||
| - | 1C9V is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus] with CL as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Hydrolase Hydrolase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.10.130.10 3.10.130.10] Full crystallographic information is available from [http:// | + | 1C9V is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus] with <scene name='pdbligand=CL:'>CL</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Hydrolase Hydrolase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.10.130.10 3.10.130.10] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1C9V OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Park, C.]] | [[Category: Park, C.]] | ||
| - | [[Category: Raines, R | + | [[Category: Raines, R T.]] |
| - | [[Category: Schultz, L | + | [[Category: Schultz, L W.]] |
[[Category: CL]] | [[Category: CL]] | ||
[[Category: antiparallel beta sheet]] | [[Category: antiparallel beta sheet]] | ||
[[Category: hydrolase]] | [[Category: hydrolase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:03:59 2008'' |
Revision as of 10:04, 21 February 2008
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H12A VARIANT OF RIBONUCLEASE A
Overview
His12 and His119 are critical for catalysis of RNA cleavage by ribonuclease A (RNase A). Substitution of either residue with an alanine decreases the value of k(cat)/K(M) by more than 10(4)-fold. His12 and His119 are proximal to the scissile phosphoryl group of an RNA substrate in enzyme-substrate complexes. Here, the role of these active site histidines in RNA binding was investigated by monitoring the effect of mutagenesis and pH on the stability of enzyme-nucleic acid complexes. X-ray diffraction analysis of the H12A and H119A variants at a resolution of 1.7 and 1.8 A, respectively, shows that the amino acid substitutions do not perturb the overall structure of the variants. Isothermal titration calorimetric studies on the complexation of wild-type RNase A and the variants with 3'-UMP at pH 6.0 show that His12 and His119 contribute 1.4 and 1.1 kcal/mol to complex stability, respectively. Determination of the stability of the complex of wild-type RNase A and 6-carboxyfluorescein approximately d(AUAA) at varying pHs by fluorescence anisotropy shows that the stability increases by 2.4 kcal/mol as the pH decreases from 8.0 to 4.0. At pH 4.0, replacing His12 with an alanine residue decreases the stability of the complex with 6-carboxyfluorescein approximately d(AUAA) by 2.3 kcal/mol. Together, these structural and thermodynamic data provide the first thorough analysis of the contribution of histidine residues to nucleic acid binding.
About this Structure
1C9V is a Single protein structure of sequence from Bos taurus with as ligand. Active as Hydrolase, with EC number 3.10.130.10 Full crystallographic information is available from OCA.
Reference
Contribution of the active site histidine residues of ribonuclease A to nucleic acid binding., Park C, Schultz LW, Raines RT, Biochemistry. 2001 Apr 24;40(16):4949-56. PMID:11305910
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