Spectral studies of conformational change at the active site of mutant O-acetylserine sulfhydrylase-A (C43S)

被引:0
|
作者
Park, JB [1 ]
Kim, SK [1 ]
Yoon, MY [1 ]
机构
[1] HANYANG UNIV,DEPT CHEM,SEOUL 133791,SOUTH KOREA
来源
关键词
mechanism; O-acetylserine sulfhydrylase; spectral study;
D O I
暂无
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The cysteine 43, potentially important in the activity of O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium, has been changed to serine. This mutant enzyme (C43S) has been studied in order to gain insight into the structural basis for the binding of inhibitor, substrate and product. UV-visible spectra of C43S exhibit the same spectral change in the presence of OAS as that observed with wild type enzyme, indicating C43S will form an alpha-aminoacrylate Schiff base intermediate. At pH 6.5, however, the deacetylase activity of C43S is much higher than wild type enzyme indicating that cysteine 43 plays a role in stabilizing the alpha-aminoacrylate intermediate. The fluorescence spectrum of C43S exhibits a ratio of emission at 340 to 502 nm of 16.9, reflecting the lower fluorescence of PLP and indicating that the orientation of cofactor and tryptophan are different from that of the wild type enzyme. The emission spectrum of C43S in the presence of OAS gives two maxima at 340 and 535 nm. The 535 nm emission is attributed to the fluorescence of the alpha-aminoacrylate intermediate. The visible circular dichroic spectrum was similar to wild type enzyme, but the negative effect observed at 530 similar to 550 nm and the molar ellipicity values for the mutant are decreased by about 50% compared to wild type enzyme. The circular dichroic and fluorescence studies suggest binding of the cofactor is less asymmetric in C43S than in the wild type enzyme.
引用
收藏
页码:32 / 37
页数:6
相关论文
共 15 条
  • [1] Kinetics and mechanism of mutant O-acetylserine sulfhydrylase-A (C43S) from Salmonella typhimurium LT-2
    Yoon, MY
    [J]. JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY, 1996, 29 (03): : 210 - 214
  • [2] PRODUCT BINDING TO THE ALPHA-CARBOXYL SUBSITE RESULTS IN A CONFORMATIONAL CHANGE AT THE ACTIVE-SITE OF O-ACETYLSERINE SULFHYDRYLASE-A - EVIDENCE FROM FLUORESCENCE SPECTROSCOPY
    MCCLURE, GD
    COOK, PF
    [J]. BIOCHEMISTRY, 1994, 33 (07) : 1674 - 1683
  • [3] ACID-BASE CHEMICAL MECHANISM OF O-ACETYLSERINE SULFHYDRYLASE-A AND SULFHYDRYLASE-B FROM PH STUDIES
    TAI, CH
    NALABOLU, SR
    SIMMONS, JW
    JACOBSON, TM
    COOK, PF
    [J]. BIOCHEMISTRY, 1995, 34 (38) : 12311 - 12322
  • [4] Effect of mutation of lysine-120, located at the entry to the active site of O-acetylserine sulfhydrylase-A from Salmonella typhimurium
    Tai, Chia-Hui
    Rabeh, Wael M.
    Guan, Rong
    Schnackerz, Klaus D.
    Cook, Paul F.
    [J]. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 2008, 1784 (04): : 629 - 637
  • [5] Interaction of serine acetyltransferase with O-acetylserine sulfhydrylase active site:: Evidence from fluorescence spectroscopy
    Campanini, B
    Speroni, F
    Salsi, E
    Cook, PF
    Roderick, SL
    Huang, B
    Bettati, S
    Mozzarelli, A
    [J]. PROTEIN SCIENCE, 2005, 14 (08) : 2115 - 2124
  • [6] Ligand binding induces a large conformational change in O-acetylserine sulfhydrylase from Salmonella typhimurium
    Burkhard, P
    Tai, CH
    Ristroph, CM
    Cook, PF
    Jansonius, JN
    [J]. JOURNAL OF MOLECULAR BIOLOGY, 1999, 291 (04) : 941 - 953
  • [7] The active site of O-acetylserine sulfhydrylase is the anchor point for bienzyme complex formation with serine acetyltransferase
    Huang, B
    Vetting, MW
    Roderick, SL
    [J]. JOURNAL OF BACTERIOLOGY, 2005, 187 (09) : 3201 - 3205
  • [8] Cysteine 42 is important for maintaining an integral active site for O-Acetylserine sulfhydrylase resulting in the stabilization of the α-aminoacrylate intermediate
    Tai, CH
    Yoon, MY
    Kim, SK
    Rege, VD
    Nalabolu, SR
    Kredich, NM
    Schnackerz, KD
    Cook, PF
    [J]. BIOCHEMISTRY, 1998, 37 (30) : 10597 - 10604
  • [9] Fine tuning of the active site modulates specificity in the interaction of O-acetylserine sulfhydrylase isozymes with serine acetyltransferase
    Spyrakis, Francesca
    Felici, Paolo
    Bayden, Alexander S.
    Salsi, Enea
    Miggiano, Riccardo
    Kellogg, Glen E.
    Cozzini, Pietro
    Cook, Paul F.
    Mozzarelli, Andrea
    Campanini, Barbara
    [J]. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 2013, 1834 (01): : 169 - 181
  • [10] SEQUENCE AROUND THE ACTIVE-SITE LYSINE AND STEREOCHEMISTRY OF REDUCTION OF O-ACETYLSERINE SULFHYDRYLASE FROM SALMONELLA-TYPHIMURIUM
    TAI, CH
    COOK, PF
    [J]. FASEB JOURNAL, 1991, 5 (04): : A441 - A441