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KMID : 0613820150250050487
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Journal of Life Science
2015 Volume.25 No. 5 p.487 ~ p.495
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Inhibition and Chemical Mechanism of Protocatechuate 3,4-dioxygenase from Pseudomonas pseudoalcaligenes KF707
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Kang Tae-Kyeong
Kim Sang-Ho
Jung Mi-Ja
Cho Yong-Kweon
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Abstract
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We carried out pH stability, chemical inhibition, chemical modification, and pH-dependent kinetic parameter assessments to further characterize protocatechuate 3,4-dioxygenase from Pseudomonas pseudoalcaligenes KF707. Protocatechuate 3,4-dioxygenase was stable in the pH range of 4.5~10.5. L-ascorbate and glutathione were competitive inhibitors with Kis values of 0.17 mM and 0.86 mM, respectively. DL-dithiothreitol was a noncompetitive inhibitor with a Kis value of 1.57 mM and a Kii value of 8.08 mM. Potassium cyanide, p-hydroxybenzoate, and sodium azide showed a noncompetitive inhibition pattern with Kis values of 55.7 mM, 0.22 mM, and 15.64 mM, and Kii values of 94.1 mM, 8.08 mM, and 662.64 mM, respectively. FeCl2 was the best competitive inhibitor with a Kis value of 29 μM. FeCl3, MnCl2, CoCl2, and AlCl3 were also competitive inhibitors with Kis values of 1.21 mM, 0.85 mM, 3.98 mM, and 0.21 mM, respectively. Other metal ions showed noncompetitive inhibition patterns. The pH-dependent kinetic parameter data showed that there may be at least two catalytic groups with pK values of 6.2 and 9.4 and two binding groups with pK values of 5.5 and 9.0. Lysine, cysteine, tyrosine, carboxyl, and histidine were modified by their own specific chemical modifiers, indicating that they are involved in substrate binding and catalysis.
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KEYWORD
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Chemical modification, pH-dependent kinetic parameters, protocatechuate 3, 4-dioxygenase, Pseudomonas pseudoalcaligenes KF707
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