Pseudomonas pseudoalcaligenes KF707에서 유래한 protocatechuate 3,4-dioxygenase 의 저해 및 화학적 메커니즘

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강태경;김상호;정미자;조용권
Kang, Taekyeong;Kim, Sang Ho;Jung, Mi Ja;Cho, Yong Kweon

  • 투고 : 2015.01.27
  • 심사 : 2015.03.21
  • 발행 : 2015.05.30

초록

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. $FeCl_{2}$는 Kis가 $29{\mu}M$로 가장 우수한 경쟁적 저해제였으며, $FeCl_{3}$, $MnCl_{2}$, $CoCl_{2}$, $HgCl_{2}$, $AlCl_{3}$도 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.

키워드

Chemical modification;pH-dependent kinetic parameters;protocatechuate 3;4-dioxygenase;Pseudomonas pseudoalcaligenes KF707

참고문헌

  1. Sim, H. W., Jung, M. J. and Cho, Y. K. 2013. Purification and Characterization of Protocatechuate 3,4-dioxygenase from Pseudomonas pseudoalcaligenes KF707. J. Kor. Soc. App. Biol. Chem. 56, 401-408. https://doi.org/10.1007/s13765-013-3080-2
  2. True, A. E., Orville, A. M., Pearce, L. L., Lipscomb, J. D. and Que, L. Jr. 1990. An EXAFS study of the interaction of substrate with the ferric active site of protocatechuate 3,4-dioxygenase. Biochemistry 29, 10847-10854. https://doi.org/10.1021/bi00500a019
  3. Valley, M. P., Brown, C. K., Burk, D. L., Vetting, M. W., Ohlendorf, D. H. and Lipscomb, J. D. 2005. Roles of the equatorial tyrosyl iron ligand of protocatechuate 3,4-dioxygenase in catalysis. Biochemistry 44, 11024-11039. https://doi.org/10.1021/bi050902i
  4. Vetting, M. W., D'argenio, D. A., Ornston, L. N. and Ohlendorf, D. H. 2000. Structure of acinetobacter strain adp1 protocatechuate 3,4-dioxygenase at 2.2 Å resolution: implications for the mechanism of an intradiol dioxygenase. Biochemistry 39, 7943-7955. https://doi.org/10.1021/bi000151e
  5. Whitaker, J. R. 1995. Polyphenol oxidase, pp. 271-307. In: Wong, D. W. S. (ed), Food enzymes structure and mechanism. Chapman & Hall: New York, NY, USA.
  6. Lundblad, R. L. and Noyes, C. M. 1984a. Chemical reagents for protein modification. Vol I, pp. 1-166, CRC Press: Boca Raton, FL, USA.
  7. Lundblad, R. L. and Noyes, C. M. 1984b. Chemical reagents for protein modification. Vol II, pp. 1-169, CRC Press: Boca Raton, FL, USA.
  8. Mapson, L. W. and Tomalin, A. W. 1961. Preservation of peeled potato. Ⅲ. The inactivation of phenolase by heat. J. Sci. Food Agr. 12, 54-58. https://doi.org/10.1002/jsfa.2740120110
  9. Orville, A. M., Lipscomb, J. D. and Ohlendorf, D. H. 1997a. Crystal structures of substrate and substrate analog complexes of protocatechuate 3,4-dioxygenase: endogenous Fe3+ ligand displacement in response to substrate binding. Biochemistry 36, 10052-10066. https://doi.org/10.1021/bi970469f
  10. Martinez, M. V. and Whitaker, J. R. 1995. The biochemistry and control of enzymatic browning. Trends Food Sci. Technol. 6, 195-200. https://doi.org/10.1016/S0924-2244(00)89054-8
  11. Montgomery, M. W. 1983. Cysteine as an inhibitor of browning in pear juice concentrate. J. Food Sci. 48, 951-952. https://doi.org/10.1111/j.1365-2621.1983.tb14937.x
  12. Ohlendorf, D. H., Orville, A. M. and Lipscomb, J. D. 1994. Structure of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa at 2.15 Å resolution. J. Mol. Biol. 244, 586-608. https://doi.org/10.1006/jmbi.1994.1754
  13. Orville, A. M., Elango, N. E., Lipscomb, J. D. and Ohlendorf, D. H. 1997b. Structures of competitive inhibitor complexes of protocatechuate 3,4-dioxygenase: multiple exogenous ligand binding orientations within the active site. Biochemistry 36, 10039-10051. https://doi.org/10.1021/bi970468n
  14. Ponting, J. D., Jackson, R. and Watters, G. 1971. Refrigerated apple slices. Effects of pH, sulfites and calcium on texture. J. Food Sci. 36, 349-350. https://doi.org/10.1111/j.1365-2621.1971.tb04059.x
  15. Price, N. C. and Stevens, L. 1998. Fundamentals of Enzymology. Oxford University Press: New York, NY, USA.
  16. Frazee, R. W., Orville, A. M., Dolbeare, K. B., Yu, H., Ohlendorf, D. H. and Lipscomb, J. D. 1998. The axial tyrosinate Fe3+ ligand in protocatechuate 3,4-dioxygenase influences substrate binding and product release: evidence for new reaction cycle intermediates. Biochemistry 37, 2131-2144. https://doi.org/10.1021/bi972047b
  17. Friedman, M. 1996. Food browning and its prevention: an overview. J. Agric. Food Chem. 44, 631-653. https://doi.org/10.1021/jf950394r
  18. Kelly, S. H. and Finkle, B. J. 1969. Action of a ring-cleaving oxygenase in preventing oxidase darkening of apple juice. J. Sci. Food Agr. 20, 629-632. https://doi.org/10.1002/jsfa.2740201015
  19. Fujisawa, H., Hiromi, K., Uyeda, M., Okuno, S., Nozaki, M. and Hayaishi, O. 1972. Protocatechuate 3,4 dioxygenase III. An oxygenated form of the enzyme as reaction intermediate. J. Biol. Chem. 247, 4422-4428.
  20. Joslyn, M. A. and Ponting, J. D. 1951. Enzyme-catalyzed oxidative browning of fruit products. Adv. Food Res. 3, 1-44. https://doi.org/10.1016/S0065-2628(08)60258-X
  21. Kahn, V. 1985. Effect of proteins, protein hydrolyzates and amino acids on o-dihydroxyphenolase activity of polyphenol oxidase of mushroom, avocado, and banana. J. Food Sci. 50, 111-115.
  22. Kurahashi, T., Oda, K., Sugimoto, M., Ogura, T. and Fujii, H. 2006. Trigonal-bipyramidal geometry induced by an external water ligand in a sterically hindered iron salen complex, related to the active site of protocatechuate 3,4- dioxygenase. Inorg. Chem. 45, 7709-7721. https://doi.org/10.1021/ic060650p
  23. Lim, J. C., Gruschus, J. M., Kim, G., Berlett, B. S., Tjandra, N. and Levine, R. L. 2012. A low pKa cysteine at the active site of mouse methionine sulfoxide reductase A. J. Biol. Chem. 287, 25596-601. https://doi.org/10.1074/jbc.M112.369116
  24. Lukes, B., O'brien, T. J. and Scanlan, R. A. 1980. Residual sulfur dioxide in finished malt: Colorimetric determination and relation to N-nitrosodimethylamine. Am. Soc. Brew. Chem. J. 38, 146-148.
  25. Bedrosian, K., Nelson, A. I. and Seinberg, M. P. 1959. Effect of borates and other inhibitors on enzymatic browning in apple tissues. Food Technol. 13, 722-726.
  26. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  27. Cho, Y. K. and Cook, P. F. 1988. Inactivation of pyrophosphate- dependent phosphofructokinase from Propionibacterium freudenreichii by pyridoxal 5’-phosphate. J. Biol. Chem. 263, 5135-5140.
  28. Cleland, W. W. 1979. Statistical analysis of enzyme kinetic data. Meth. Enzymol. 63, 103-138. https://doi.org/10.1016/0076-6879(79)63008-2
  29. Dagleys, S. 1984. Microbial degradation of aromatic compounds. Devel. Ind. Microbiol. 25, 53-65.
  30. Davis, M. L., Wasinger, E. C., Westre, T. E., Zaleski, J. M., Orville, A. M., Lipscomb, J. D., Hedman, B., Hodgson, K. O. and Solomon, E. I. 1999. Spectroscopic investigation of reduced protocatechuate 3,4-dioxygenase: charge-induced alterations in the active site iron coordination environment. Inorg. Chem. 38, 3676-3683. https://doi.org/10.1021/ic981464p
  31. Durham, D. R., Stirling, L. A., Ornston, L. N. and Perry, J. J. 1980. Intergeneric evolutionary homology revealed by the study of protocatechuate 3,4-dioxygenase from Azotobacter vinelandii. Biochemistry 19, 149-155. https://doi.org/10.1021/bi00542a023
  32. Elgren, T. E., Orville, A. M., Kelly, K. A., Lipscomb, J. D., Ohlendorf, D. H. and Que, L. Jr. 1997. Crystal structure and resonance raman studies of protocatechuate 3,4-dioxygenase complexed with 3,4-dihydroxyphenylacetate. Biochemistry 36, 11504-11513. https://doi.org/10.1021/bi970691k
  33. Engel, P. C. 1996. Enzymology LabFax. pp. 175-190, Academic Press: San Diego, CA, USA.
  34. Ashie, I. N. A., Simpson, B. K. and Smith, J. P. 1996. Mechanisms for controlling enzymatic reactions in foods. Crit. Rev. Food Sci. Nutr. 36, 1-30.