Unusual pK a of the carboxylate at the putative catalytic position of the thermophilic F1‐ATPase β subunit determined by 13C‐NMR

FEBS Letters

Akutsu, Hideo²  Tozawa, Kaeko²  Ohbuchi, Hiroshi²  Yagi, Hiromasa²  Amano, Toyoki¹  Matsui, Tadashi¹  Yoshida, Masasuke¹ 

[1] Research Laboratory of Resources Utilization, R-1, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226, Japan;Department of Bioengineering, Faculty of Engineering, Yokohama National University, 156 Tokiwadai, Hodogaya-ku, Yokohama 240, Japan

关键词: F1-ATPase;    pK a;    Carboxymethylation;    Glutamic acid;    Specific 13C labeling;    13C-NMR;    (Thermophilic Bacillus);    AMP-PNP;    5′-adenylyl imidodiphosphate;    α(C193S/W463F);    mutant α subunit in which Cys-193 and Trp-463 are substituted by serine and phenylalanine;    respectively;    β(E190C);    mutant β subunit in which Glu-190 is substituted by cysteine;    CmCys;    S-carboxymethylcysteine;    Cmβ;    S-carboxymethylated at Cys-190 of β(E190C);    DCCD;    N;    N′-dicyclohexylcarbodiimide;    DTNB;    5;    5′-dithiobis-2-nitrobenzoic acid;    IAA;    monoiodoacetic acid;    Mg·AMP-PNP;    an equimolar mixture of MgCl2 and AMP-PNP;    TF1;    F1-ATPase from thermophilic Bacillus strain PS-3;    TF1β;    β subunit of TF1-ATPase;   

DOI  :  10.1016/S0014-5793(96)01155-6

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Glutamic acid-190 in the β subunit of F₁-ATPase from thermophilic Bacillus PS-3 (TF₁) was reporte to be essential for the ATPase activity. The mutant TF₁β subunit in which Glu-190 had been substituted by cysteine was carboxymethylated with ¹³C-labeled monoiodoacetic acid. The pK _a value of the carboxymethylene group at the 190 position was determined as 5.6 ± 0.4 by ¹³C-NMR. On the basis of this value, the pK _a of the carboxylate of Glu-190 of the TF₁β subunit was estimated to be 6.8 ± 0.5. The unusually high pK _a could play a role in the catalytic mechanism of F₁-ATPase.

【 授权许可】

Unknown