Reconstitution of apo‐porphobilinogen deaminase: Structural changes induced by cofactor binding

FEBS Letters

Scott, A.Ian¹  Roessner, Charles A.¹  Clemens, Karen R.¹  Santander, Patricio J.¹  Stolowich, Neal J.¹  Gonzalez, Mario D.¹ 

[1] Center for Biological NMR, Department of Chemistry, Texas A&M University, College Station, TX 77843, USA

关键词: Porphobilinogen deaminase;    NMR spectroscopy;    13C-;    Dipyrromethane cofactor;    Enzyme conformation;    Reconstitution;    (E. coli);    PBG;    porphobilinogen;    ALA;    5-aminolevulinic acid;    uro'gen;    uroporphyrinogen;    PE buffer;    phosphate-EDTA buffer (100 mM KH2PO4;    2 mM EDTA adjusted to pH 8.0 with NaOH);    PAGE;    polyacrylamide gel electrophoresis;    ES1;    ES2;    ES3;    and ES4;    enzyme-substrate complexes in which PBG deaminase with the covalently attached dipyrromethane cofactor (E) has reacted with 1;    2;    3;    or 4 mol PBG;   

DOI  :  10.1016/0014-5793(89)80493-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Expression of porphobilinogen deaminase in a hemB ⁻ strain of E. coli has permitted the isolation of the apoenzyme, i.e. deaminase lacking the porphobilinogen-derived dipyrromethane cofactor. Incubation of purified apoenzyme with porphobilinogen resulted in reconstitution of the covalently attached dipyrromethane cofactor, indicating no additional cofactors or enzymes are required for biosynthesis of holoenzyme. Electrophoretic and ¹³C-NMR spectroscopic analyses demonstrate that the apoenzyme exists in a conformationally unstable form which is converted to a highly stable tertiary structure on covalent attachment of the dipyrromethane cofactor.

【 授权许可】

Unknown