1fun
From Proteopedia
(New page: 200px<br /> <applet load="1fun" size="450" color="white" frame="true" align="right" spinBox="true" caption="1fun, resolution 2.85Å" /> '''SUPEROXIDE DISMUTAS...) |
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| - | [[Image:1fun.gif|left|200px]]<br /> | + | [[Image:1fun.gif|left|200px]]<br /><applet load="1fun" size="350" color="white" frame="true" align="right" spinBox="true" |
| - | <applet load="1fun" size=" | + | |
caption="1fun, resolution 2.85Å" /> | caption="1fun, resolution 2.85Å" /> | ||
'''SUPEROXIDE DISMUTASE MUTANT WITH LYS 136 REPLACED BY GLU, CYS 6 REPLACED BY ALA AND CYS 111 REPLACED BY SER (K136E, C6A, C111S)'''<br /> | '''SUPEROXIDE DISMUTASE MUTANT WITH LYS 136 REPLACED BY GLU, CYS 6 REPLACED BY ALA AND CYS 111 REPLACED BY SER (K136E, C6A, C111S)'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Key charged residues in Cu,Zn superoxide dismutase (Cu,Zn SOD) promote | + | Key charged residues in Cu,Zn superoxide dismutase (Cu,Zn SOD) promote electrostatic steering of the superoxide substrate to the active site Cu ion, resulting in dismutation of superoxide to oxygen and hydrogen peroxide, Lys-136, along with the adjacent residues Glu-132 and Glu-133, forms a proposed electrostatic triad contributing to substrate recognition. Human Cu,Zn SODs with single-site replacements of Lys-136 by Arg,Ala, Gln, or Glu or with a triple-site substitution (Glu-132 and Glu-133 to Gln and Lys-136 to Ala) were made to test hypotheses regarding contributions of these residues to Cu,Zn SOD activity. The structural effects of these mutations were modeled computationally and validated by the X-ray crystallographic structure determination of Cu,Zn SOD having the Lys-136-to-Glu replacement. Brownian dynamics simulations and multiple-site titration calculations predicted mutant reaction rates as well as ionic strength and pH effects measured by pulse-radiolytic experiments. Lys-136-to-Glu charge reversal decreased dismutation activity 50% from 2.2 x 10(9) to 1.2 x 10(9) M-1 s-1 due to repulsion of negatively charged superoxide, whereas charge-neutralizing substitutions (Lys-136 to Gln or Ala) had a less dramatic influence. In contrast, the triple-mutant Cu,Zn SOD (all three charges in the electrostatic triad neutralized) surprisingly doubled the reaction rate compared with wild-type enzyme but introduced phosphate inhibition. Computational and experimental reaction rates decreased with increasing ionic strength in all of the Lys-136 mutants, with charge reversal having a more pronounced effect than charge neutralization, implying that local electrostatic effects still govern the dismutation rates. Multiple-site titration analysis showed that deprotonation events throughout the enzyme are likely responsible for the gradual decrease in SOD activity above pH 9.5 and predicted a pKa value of 11.7 for Lys-136. Overall, Lys-136 and Glu-132 make comparable contributions to substrate recognition but are less critical to enzyme function than Arg-143, which is both mechanistically and electrostatically essential. Thus, the sequence-conserved residues of this electrostatic triad are evidently important solely for their electrostatic properties, which maintain the high catalytic rate and turnover of Cu,Zn SOD while simultaneously providing specificity by selecting against binding by other anions. |
==Disease== | ==Disease== | ||
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==About this Structure== | ==About this Structure== | ||
| - | 1FUN is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with CU, ZN and SO4 as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Superoxide_dismutase Superoxide dismutase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.15.1.1 1.15.1.1] Full crystallographic information is available from [http:// | + | 1FUN is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with <scene name='pdbligand=CU:'>CU</scene>, <scene name='pdbligand=ZN:'>ZN</scene> and <scene name='pdbligand=SO4:'>SO4</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Superoxide_dismutase Superoxide dismutase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.15.1.1 1.15.1.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1FUN OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Superoxide dismutase]] | [[Category: Superoxide dismutase]] | ||
| - | [[Category: Getzoff, E | + | [[Category: Getzoff, E D.]] |
| - | [[Category: Lo, T | + | [[Category: Lo, T P.]] |
| - | [[Category: Tainer, J | + | [[Category: Tainer, J A.]] |
[[Category: CU]] | [[Category: CU]] | ||
[[Category: SO4]] | [[Category: SO4]] | ||
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[[Category: superoxide acceptor]] | [[Category: superoxide acceptor]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:42:51 2008'' |
Revision as of 10:42, 21 February 2008
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SUPEROXIDE DISMUTASE MUTANT WITH LYS 136 REPLACED BY GLU, CYS 6 REPLACED BY ALA AND CYS 111 REPLACED BY SER (K136E, C6A, C111S)
Contents |
Overview
Key charged residues in Cu,Zn superoxide dismutase (Cu,Zn SOD) promote electrostatic steering of the superoxide substrate to the active site Cu ion, resulting in dismutation of superoxide to oxygen and hydrogen peroxide, Lys-136, along with the adjacent residues Glu-132 and Glu-133, forms a proposed electrostatic triad contributing to substrate recognition. Human Cu,Zn SODs with single-site replacements of Lys-136 by Arg,Ala, Gln, or Glu or with a triple-site substitution (Glu-132 and Glu-133 to Gln and Lys-136 to Ala) were made to test hypotheses regarding contributions of these residues to Cu,Zn SOD activity. The structural effects of these mutations were modeled computationally and validated by the X-ray crystallographic structure determination of Cu,Zn SOD having the Lys-136-to-Glu replacement. Brownian dynamics simulations and multiple-site titration calculations predicted mutant reaction rates as well as ionic strength and pH effects measured by pulse-radiolytic experiments. Lys-136-to-Glu charge reversal decreased dismutation activity 50% from 2.2 x 10(9) to 1.2 x 10(9) M-1 s-1 due to repulsion of negatively charged superoxide, whereas charge-neutralizing substitutions (Lys-136 to Gln or Ala) had a less dramatic influence. In contrast, the triple-mutant Cu,Zn SOD (all three charges in the electrostatic triad neutralized) surprisingly doubled the reaction rate compared with wild-type enzyme but introduced phosphate inhibition. Computational and experimental reaction rates decreased with increasing ionic strength in all of the Lys-136 mutants, with charge reversal having a more pronounced effect than charge neutralization, implying that local electrostatic effects still govern the dismutation rates. Multiple-site titration analysis showed that deprotonation events throughout the enzyme are likely responsible for the gradual decrease in SOD activity above pH 9.5 and predicted a pKa value of 11.7 for Lys-136. Overall, Lys-136 and Glu-132 make comparable contributions to substrate recognition but are less critical to enzyme function than Arg-143, which is both mechanistically and electrostatically essential. Thus, the sequence-conserved residues of this electrostatic triad are evidently important solely for their electrostatic properties, which maintain the high catalytic rate and turnover of Cu,Zn SOD while simultaneously providing specificity by selecting against binding by other anions.
Disease
Known disease associated with this structure: Amyotrophic lateral sclerosis, due to SOD1 deficiency OMIM:[147450]
About this Structure
1FUN is a Single protein structure of sequence from Homo sapiens with , and as ligands. Active as Superoxide dismutase, with EC number 1.15.1.1 Full crystallographic information is available from OCA.
Reference
Computational, pulse-radiolytic, and structural investigations of lysine-136 and its role in the electrostatic triad of human Cu,Zn superoxide dismutase., Fisher CL, Cabelli DE, Hallewell RA, Beroza P, Lo TP, Getzoff ED, Tainer JA, Proteins. 1997 Sep;29(1):103-12. PMID:9294870
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