Leuconostoc mesenteroides에서 分離한 Glucose-6-phosphate Dehydrogenase의 特性

Characteristics of Glucose-6-phosphate Dehydrogenase from Leuconostoc mesenteroides

  • 변시명 (韓國科學院 生物工學科) ;
  • 최양도 (韓國科學院 生物工學科) ;
  • 한문희 (韓國科學技術硏究所 應用生化學硏究室)
  • Byun Si Myung (Department of Biological Science Korea Advanced Institute of Science) ;
  • Yang Do Choi (Department of Biological Science Korea Advanced Institute of Science) ;
  • Moon H. Han (Applied Biochemistry Laboratory, Korea Institute of Science and Technology)
  • 발행 : 1979.08.30

초록

저자들은 Cibacron Blue F3G-A Separose 컬럼 어피니티크로마토그래피에 의하여 GIn-cose-6-phosphate dehydrogenase를 Leuconostoc mesenteroides로부터 순수 분리한 바 있다. 이 효소를 사용하여 효소특성을 조사한 결과 분자량은 Sephadex G-200 컬럼에 의해 112,000이었으며 최적온도는 50$^{\circ}$, 활성화에너 지는 8.36kcal/mole 불활성화에너지는 -58.2kcal/mole로 나타났다. $NADP^+$를 조효소로 사용하였을때 최적 pH7.8에서 K_{G6p}:76.9${\mu}$M, ${\alpha}K_{NADP}:\;7.46{\mu}M,\;{\alpha}KNNADP:\;7.l4{\mu}M$이었으며 같은 조건에서 $NAD^+$를 조효소로 사용하였을때 $K_{G6P}:\;53.65{\mu}M,\;K_{NAD}:\;115.2{\mu}M\;{\alpha}K_{NAD}:\;707.2{\mu}M$이었다. 따라서 $NADP^+$$NAD^+$를 조효소로 사용한 경우에 있어서 ${\alpha}$ 값은 각각 1과 6으로 나타났다. pH변화에 따른 반응속도상수의 변화에 의하면 $NAD^+$를 조효소로 하였을때 최적 pH는 7.8 이었고 pKa가 7.2인 활성기와 ${\mu}Kb$가 9.0∼9.6인 활성기가 효소와 기질의 상호작용에 관여함을 알았다. 이중 pKa 7.2인 활성기를 밝히기 위하여 효소를 광산화와 carboxymethylation을 시킨결과 histidine의 imidazole기임을 알수 있있다.

Glucose 6-phosphate dehydrogenase of Leuconostoc mesenteroides which was purifid by an affinity chromatography was studied on the characterization, kinetics and chemical modification. The apparent molecular weight of the enzyme was 112,000 by the gel filtration method of Sephadex G-200 column. The optimum temperature of $NAD^+$-linked reation was 50$^{circ}C$ and the activation energy and the heat of inactivation were 8.36 kcal/mole and -58.2kcal/mole, respectively. The steady state kinetic study showed KG6P, Kemp, and CX KNADP to be 76.9 PM, 7.46${\mu}M$ and 7.14 ${\mu}M$, respectively, and KGGP, KNAD,and aKNm to be 53.7${\mu}M$, 115.2${\mu}M$ and 702.2${\mu}M$ for the $NAD^+$-linked reaction at pH 7.8, optimum pH. The pH dependent kinetic constants suggested that the two ionizing groups whose pKa is 7.2 .and pKb is 9.0-9.6 were involved in the enzyme-substrate interaction. Evidence by photooxidation and carboxymethylation of the enzyme suggested that the imidazole group of histidine with pKa group may participate in the catalytic site.

키워드

참고문헌

  1. Biochem. Biophys. Acta v.451 P. A. Aitchison;J.M. Aitchison;M. M. Yeoman
  2. Biochem. Bioahys. Res. Comm. v.6 H. R. Levy
  3. J. Biol. Chem. v.241 H. R. Levy;R. R. Raineri;B. H. Nevaldine
  4. J. Biol. Chem. v.236 P. A. Marks;A. Szeinberg;J. Banks
  5. Biochem. Z. v.287 O. Warburg;W. Chistian
  6. Hoppe-Seyler's Z. Physiol. Chem. v.350 G. F. Domagk;R. Chilla;W. Domschke;H. J. Engel;N. Sorensen
  7. J. Bacteriol. v.66 R. D. DeMoss;I. C. Gunsalus;R. C. Bard
  8. Biochem. v.6 C. Olive;H. R. Levy
  9. J. Biol. Chem. v.246 C. Olive;H. R. Levy
  10. Eur. J. Biochem. v.50 M. Milhausen;H. R. Levy
  11. Arch. Biochem. Biopys. v.177 T. H. Grove;A. Ishaque;H.R. Levy
  12. Methods in Enzymolgy v.1 R. D. DeMoss;S. P. Colowick(ed.);N. O. Kaplan(ed.)
  13. Biochem. Biophys. Res. Comm. v.59 A. IshaqueM. Milhausen;R.H. Levy
  14. J. Biol. Chem. v.246 C. Olive;M. E. Geroch;H. R. Levy
  15. Korean J. Appl. Microbiol. Bioeng. v.5 K. E. Lee;T. H. Chung;T. I. Mheen;M. H. Han
  16. Submitted to J. Biochem. Y. D. Choi;S.M. Byun;M.H. Han
  17. Biochem. J. v.91 P. Andrews
  18. Enzyme(2nd Ed) M. Dixon;E. C. Webb
  19. Chemical Modification of Proteins G. E. Means;R. E. Feeney
  20. Methods in Enzymology v.11 F. R. N. Gurd;C.H. W. Hirs(ed.)
  21. J. Theor. Biol. v.35 M. H. Han
  22. J. Biol. Chem. v.238 A. C. Chung;R. G. Langdon
  23. J. Biol. Chem. v.237 H. N. Kirkman;E. M. Hendrickson
  24. Biochem. J. v.96 P. Andrews
  25. Arch. Biochem. Biophys. v.123 J.S. Bellin;C. A. Yankas