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

[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.

[学科分类] 生物化学/生物物理

[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