Inhibition and Chemical Mechanism of Protocatechuate 3,4-dioxygenase from Pseudomonas pseudoalcaligenes KF707

국문 초록 (Abstract)

Pseudomonas pseudoalcaligenes KF707에서 정제한 protocatechuate 3,4-dioxygenase의 특징을 조사하기 위하여 pH안정성, 화학적 저해, 화학적 수식과 pH의존성 반응 상수에 대한 실험을 수행하였다. 이 효소는 pH 4.5~10.7에서 안정하였다. L-ascorbate와 glutathione은 Kis가 각각 0.17 mM과 0.86 mM인 경쟁적 저해제였으며, DL-dithiothreitol은 Kis 1.57 mM 및 Kii 8.08 mM의 비경쟁적 저해패턴을 나타내었다. Potassium cyanide, p-hydroxybenzoate 및 sodium azide는 Kis가 각각 55.7 mM, 0.22 mM 및15.64 mM이었으며, Kii는 각각94.1 mM, 8.08
mM, 및 662.64 mM인 비경쟁적 저해패턴을 나타내었다. FeCl2는 Kis가 29 μM로 가장 우수한 경쟁적 저해제였으며, FeCl3, MnCl2, CoCl2, HgCl2, AlCl3도 각각 Kis가 1.21 mM, 0.85 mM, 3.98 mM, 0.17 mM 및 0.21 mM인 경쟁적 저해패턴을 보였다. 한편, 다른 금속이온들은 비경쟁적 저해패턴을 나타내었다. pH의존성 반응상수의 실험결과로부터 pK 6.2와 9.4의 촉매부위와 pK 5.5와 9.0의 결합부위가 존재함을 알 수 있었다. Lysine, cysteine, tyrosine, carboxyl과 histidine은 각각의 고유한 화학적 수식제에 의해 수식되었는데, 이는 이들 잔기들이 결합과 촉매에 관여한다는 것을 나타낸다. 위 결과를 토대로 화학적 메커니즘을 제시한다.

다국어 초록 (Multilingual Abstract)

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.

목차 (Table of Contents)

• Introduction
• Materials and Methods
• Results and Discussion
• References
• 초록

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