13C Nuclear magnetic resonance studies of binding of thiocyanate to lactoperoxidase and horseradish peroxidase heme enzymes
IR@NISCAIR: CSIR-NISCAIR, New Delhi - ONLINE PERIODICALS REPOSITORY (NOPR)
View Archive Info| Field | Value | |
| Title |
13C Nuclear magnetic resonance studies of binding of thiocyanate to lactoperoxidase and horseradish peroxidase heme enzymes
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| Creator |
Modi, Sandeep
Behere, Digambar V. Mitra, Samaresh |
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| Description |
301-311
Interaction of thiocyanate with lactoperoxidase (LPO) and horseradish peroxidase (HRP) has been investigated by relaxation rate measurements (at 125.77 MHz) of <sup>13</sup>C resonance of thiocyanate carbon. The apparent dissociation constant (<em>K</em><sub>D</sub>) for thiocyanate binding to LPO at <em>p</em>H = 6.1 and to HRP at <em>p</em>H = 4.0 has been deduced to be 85 m<em>M </em>and 160 m<em>M </em>respectively from the relaxation rate measurements. The <em>p</em>H dependence of <em>K</em><sub>D</sub> and <sup>13</sup>C resonance line-width of thiocyanate has been used to calculate <em>pK</em><sub>a</sub> value of amino acid residue on these enzymes where the thiocyanate is shown to be binding. From the <em>p</em>H dependence of <em>K</em><sub>D</sub> and <sup>13</sup>C resonance line-width, it is observed that thiocyanate binds to LPO and HRP only under acidic conditions (<em>p</em>H < 6.1 for LPO and <em>p</em>H < 4.0 for HRP). The binding is facilitated by protonation of an acid group on the enzyme with <em>pK</em><sub>a</sub> = 6.1 for LPO and 4.0 for HRP. The <em>p</em>H dependence of <sup>13</sup>C resonance line-width of thiocyanate as well as <em>K</em><sub>D </sub>have been quantitatively analysed on the basis of a reaction scheme in which thiocyanate in deprotonated ionic form binds to the enzyme in protonated acidic form. <em>K</em><sub>D</sub> for thiocyanate binding to the enzyme has also been evaluated in the presence of excess of exogenous substrates such as resorcinol, cyanide and iodide. The presence of cyanide (which binds to heme iron of enzyme at sixth coordination position) does not have any effect on the binding of thiocyanate, indicating that binding site of thiocyanate ion is located away from the ferric centre of these enzymes. The presence of resorcinol, has significant effect on <em>K</em><sub>D</sub> for binding of thiocyanate to LPO but it has no effect on thiocyanate binding to HRP. The <em>K </em>D in the presence of iodide however shows that iodide competes with thiocyanate for binding at the same site in both the cases. Distance of the carbon atom of bound thiocyanate ion from ferric centre has been deduced from the <sup>13</sup>C-TJ measurements and is found to be 8.4 Ǻ and 8.0 Ǻ for LPO and HRP respectively. This distance remains unchanged by the presence of cyanide ion at the sixth coordination site of the heme iron of these enzymes. Similarity in the mode of binding of iodide and thiocyanate suggests that the oxidation of thiocyanate ion by H<sub>2</sub>O<sub>2</sub>, like that of 1<sup>-</sup> by H<sub>2</sub>O<sub>2</sub> may also proceed via two-electron transfer pathway under acidic conditions. |
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| Date |
2019-03-22T05:24:52Z
2019-03-22T05:24:52Z 1990-04 |
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| Type |
Article
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| Identifier |
0975-0975(Online); 0376-4710(Print)
http://nopr.niscair.res.in/handle/123456789/46345 |
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| Language |
en_US
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| Rights |
<img src='http://nopr.niscair.res.in/image/cc-license-sml.png'> <a href='http://creativecommons.org/licenses/by-nc-nd/2.5/in' target='_blank'>CC Attribution-Noncommercial-No Derivative Works 2.5 India</a>
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| Publisher |
NISCAIR-CSIR, India
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| Source |
IJC-A Vol.29A(04) [April 1990]
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