Spatial precision of a catalytic carboxylate of F1‐ATPase β subunit probed by introducing different carboxylate‐containing side chains

FEBS Letters

Tozawa, Kaeko¹  Murakami, Hideto¹  Amano, Toyoki¹  Yoshida, Masasuke¹ 

[1] Research Laboratory of Resources Utilization, R-1, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 227, Japan

关键词: F1-ATPase;    Catalytic carboxylate residue;    Monoiodoacetic acid;    Dicyclohexylcarbodiimide;    Cax;    S-carboxymethylcysteine;    Cam;    S-carbamoylmethylcysteine;    DCCD;    N;    N′-dicyclohexylcarbodiimide;    DTNB;    5;    5′-dithiobis-2-nitrobenzoic acid;    TNP-ATP;    and TNP-ADP;    the 2′;    3′-O-(2;    4;    6-trinitrophenyl) derivatives of ATP and ADP;    respectively;    TF1;    EF1;    MF1;    and CF1;    F1-ATPase from the thermophilic Bacillus strain PS3;    Escherichia coli;    bovine heart mitochondria;    spinach chloroplasts;    respectively;   

DOI  :  10.1016/0014-5793(94)00588-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Combining mutation and chemical modification, we have introduced Asp, Gln, Cys, S-carboxymethylcysteine (Cax) and S-carbamoylmethylcysteine (Cam) into the positions of Glu¹⁹⁰ and Glu²⁰¹ of the β subunit of F₁-ATPase from the thermophilic Bacillus PS3. The steady-state ATPase activities of α₃β₃γ complexes containing these changed β subunits were 12% (E190Cax), 7% (E190D), 3% (E190Cam), <1% (E190C), <1% (E190Q), and 73% (E201D), 40% (E201Cax), 25% (E201C), 20% (E201Q), 4% (E201Cam), of that of the wild-type α₃β₃γ complex. For the complexes containing E190C or E190Q, even the ability of single-site catalysis was lost. Thus, the presence of a carboxylate at 190 (but not at 201) is absolutely required for catalysis and its spatial precision is very strict. Analysis of inactivation of the complexes by dicyclohexylcarbodiimide suggests that Glu¹⁹⁰ and Glu²⁰¹ are interacting in the F₁-ATPase.

【 授权许可】

Unknown