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트립토판 중합효소 α 소단위 잔기 치환체 Pro96→Leu의 구조 변화에 영향을 미치는 139 및 258 잔기의 치환 효과

Effect of Substituted Residue 139 and 258 on Structural Changes of Mutant Tryptophan Synthase Pro96→Leu α-Subunit

  • 이주연 (부산대학교 자연과학대학 분자생물학과) ;
  • 정재갑 (부산대학교 자연과학대학 분자생물학과) ;
  • 신혜자 (동서대학교 응용공학부 환경공학전공) ;
  • 임운기 (부산대학교 자연과학대학 분자생물학과)
  • Lee, Joo-Youn (Department of Molecular Biology, College of Natural Sciences, Pusan National University) ;
  • Jeong, Jae-Kap (Department of Molecular Biology, College of Natural Sciences, Pusan National University) ;
  • Shin, Hae-Ja (Environmental Engineering Major, Division of Applied Engineering, Dongseo University) ;
  • Lim, Woon-Ki (Department of Molecular Biology, College of Natural Sciences, Pusan National University)
  • 발행 : 2002.08.01

초록

대장균 트립토판 중합효소(tryptophan synthase) $\alpha$ 소단 위체의 96번, 139번 과 258번 자리를 돌연변이 시킨 이중 돌연변이체 P96L/F139W, P6L/F258W와 삼중 돌연변이체 P96L/F139W/F258W의 효소 활성도와 형광 분광계를 이용한 분광학적 성질을 조사하였다. 이중 돌연변이체의 효소 활성도는 야생형과 유사하나 삼중 돌연변이체는 40% 감소된 활성도를 가진다. P96L/F139W 와 P96L/F258W의 형광세기는 야생형에 비해 감소하였으나 P96L/F139W/F258W는 야생형에 비해 증가하였다. 효소 활성도와 형광 결과는 96번의 치환이 8번 알파구조와 4번 베타 구조와 4번 알파구조 사이의 loop의 안정성에 영향을 준 것을 나타낸다. 이 결과로부터 P96L/F139W/F258W는 야생형과 다른 3차 구조를 가지나 139번과 258면 주위는 더 조밀한 구조를 가지고 있다는 것을 보여준다.

Enzymatic activities and fluorescence spectroscopic properties of the double mutant proteins P96L/F139W, P96L/F258W and a triple mutant protein P96L/F139W/F258W of tryptophan synthase $\alpha$ subunit from Escherichia coli was examined to study tertiary and local structure changes around the tryptophan residues. The enzymatic activities of P96l./F139W and P96L/F258W were similar, but P96L/F139W/F258W had lower activity, as compared to wild type. The fluorescence intensities of double mutant, P96L/F139W and P96L/F258W, were decreased but that of a triple mutant, P96L/F139W/F258W, was increased when compared to wild type. The sum of the maximum fluorescence intensity (fluorescence intensity at the λ$_{max}$) for the double mutant proteins was not equal to the intensity seen in the triple mutant protein. The enzymatic activity and fluorescence data indicate that the replacement of Pro$^{96}$ longrightarrowLeu might affect on the stability of helix 8 and the loop located between strand 4 and helix4. The result suggests that the tertiary structure of triple mutant (P96L/F139W/F258W), being different from wild type, might have more compact residual structure at the vicinity of 139 and 258.8.

키워드

참고문헌

  1. J.Biol.Chem v.249 A rapid method for preparing crystalline β2 subunit of trytophan synthase of Escherichia coli in high yied Adachi,O.;L.D.Kphn;E.W.Miles
  2. J.Biol.Chem v.270 Unfolding properties of tryptophan-containing alpha-subunits of the Escherichia coli tryptophan synthase Chio,S.G.;J.K.Hardman https://doi.org/10.1074/jbc.270.47.28177
  3. J.Biol.Chem v.270 Tryptophan-containing alpha-subunits of the Escherichia coli tryptophan synthase Enzymatic and urea stability properties Chio,S.G.;S.E.O'Donnell;K.D.Sarken;J.K.Hardman
  4. Biochermistry v.9 Kinetic studies of tryptophan synthase.Interactin of substrates with β subunit Faeder,E.J.;G.G.Hammes https://doi.org/10.1021/bi00823a003
  5. Biochermistry v.22 An active proteolytic derivation of the alpha submit of tryptophan synthase.Indentificaiton of the site of cleavage and characterization of fragment. Faeder,E.J.;G.G.Hammes
  6. J.Biol.Chem v.267 Three-dimensional structure of the tryptophan synthase α2β2 multienzyme complex from Salmomella typhimurium Hyde,C.C.;S.A.Ahmed;E.A.Padian;E.W.Miles;D.R.Davies
  7. Biochermistry v.25 Synergism in folding of double mutant of the α subunit of tryptophan synthase Hurle,M.R.;C.R.Tweedy;C.R.Mattews https://doi.org/10.1021/bi00369a002
  8. J.Biol.Chem v.266 Site-directed mutagenesis of the β subunit of tryptophan synthase from Salomonella typhimurium Kayastha.A.M.;U.Sawa;S.Nagata;E.W.Milles
  9. Eur.J.Biochem v.60 The tryptophan synthase from Escherichia coli.An improved purification procedure for the alpha subunit and binding studies with substrate analogues Kirschner,K.;R.L.Wiskocil;M.Foehn;Rezeau https://doi.org/10.1111/j.1432-1033.1975.tb21030.x
  10. Thchniques in Protein Chemistry Leggett-Bailey,J.
  11. J.Bactriol v.173 Relative activities and stabilities of mutant Escherichia coli tryptophan synthase alpha subinit Lim,W.K.;H.J.Shin;D.L.Milton;J.K.Hardman
  12. Biochermistry v.21 Guanidine hydrochloride induceed unfolding of the alpha subunit of tryptophan synthase and of the two alpha proteolytic fragments :evidence for stepwise unfolding of th two alpha domains Miles,E.W.;K.Yutani;K.Ogasahara https://doi.org/10.1021/bi00540a002
  13. J.Biol.Chem v.261 In vitro mutagenesis and overexpression of the Escherichia coli trpA gene and the partial characterization of the resultant tryptophan synthase mutant alpha subunits Milton,D.L.;M.L.Napier;R.W.Muers;J.K.Hardman
  14. Proteins:Structure,Function and Genetics v.21 Affinities of phosphylated substrates for the E.coli tryptophan synthase alpha-subunit:roles of Ser-235 and helix-8' dipole Sarker,K.D.;J.K.Hardman https://doi.org/10.1002/prot.340210207
  15. Stability of Protein Pharmaceuticals v.3 In vivo pathways of degradation and strategies fot protein stabilization Wetzel,R.