Atsuko Iwamoto

A glycine-rich sequence in the catalytic site of F-type ATPase

Affinity labeling and genetic studies on the glycine-rich sequence of the β subunit ofE. coli F-type ATPase are discussed. A model of the structure of the enzyme near the γ phosphate moiety is proposed.

Catalysis and energy coupling of H+-ATPase (ATP synthase): Molecular biological approaches

The molecular biological approach has provided important information for understanding the F0F1 H(+)-ATPase. This article focuses on our recent results on the catalytic site in the beta subunit, and the roles of alpha/beta subunit interaction and amino/carboxyl terminal interaction of the gamma subunit in energy coupling. Extensive mutagenesis of the beta subunit revealed that beta Lys-155, beta Thr-156, beta Glu-181 and beta Arg-182 are essential catalytic residues. beta Glu-185 is not absolutely essential, but a carboxyl residue may be necessary at this position. A pseudo-revertant analysis positioned beta Gly-172, beta Ser-174, beta Glu-192 and beta Val-198 in the proximity of beta Gly-149. The finding of the roles of beta Gly-149, beta Lys-155, and beta Thr-156 emphasized the importance of the glycine-rich sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser, E. coli beta residues between beta Gly-149 and beta Thr-156) conserved in many nucleotide binding proteins. The A subunits of vacuolar type ATPases may have a similar catalytic mechanism because they have conserved glycine-rich and Gly-Glu-Arg (corresponding to beta Gly-180-beta Arg-182) sequences. The results of these mutational studies are consistent with the labeling of beta Lys-155 and beta Lys-201 with AP3-PL, and of beta Glu-192 with DCCD [15]. The DCCD-binding residue of a thermophilic Bacillus corresponds to beta Glu-181, an essential catalytic residue discussed above. The defective coupling of the beta Ser-174-->Phe mutant was suppressed by the second mutation alpha Arg-296-->Cys, indicating the importance of alpha/beta interaction in energy coupling. The gamma subunit, especially its amino/carboxyl interaction, seems to be essential for energy coupling between catalysis and transport judging from studies on gamma Met-23-->Lys or Arg mutation and second-site mutations which suppressed the gamma Lys-23 mutation. Thus the conserved gamma Met-23 is not absolutely essential but is located in the important region for amino/carboxyl interaction for energy coupling.

N-Ethylmaleimide-sensitive mutant (βVal-153→Cys) Escherichia coli F1-ATPase: Cross-linking of the mutant β subunit with the α subunit

A β subunit mutation, βVal‐153→Cys, in the glycine‐rich sequence (phosphate‐binding loop) of Escherichia coli F1 was constructed. Like vacuolar‐type ATPase, the mutant enzyme was inhibited by N‐ethylmaleimide (NEM) and labeled with [14C]NEM. The inhibition and labeling were prevented by ATP. m‐Maleimidobenzoyl‐N‐hydroxysuccinimide (MBS) (3 μM) almost completely inhibited the mutant enzyme, and cross‐linked one pair of α and β subunits. These results suggest that the interaction of the domain near βVal‐153 with the α subunit is essential for catalytic cooperativity of the enzyme and that βVal‐153 is within 10 Å of the α subunit.

F-type H+-ATPase: Catalysis and Proton Transport

F0F1 H+ATPase (or F-type ATPase) catalyzes ATP synthesis or hydrolysis coupling with proton translocation (for reviews, see Futai et al., 1989; Senior, 1990; Fillingame, 1990). The F-type ATPase of Escherichia coli is similar to those found in inner mitochondrial or chloroplast thylakoid membranes, and has contributed greatly to the understanding of this complicated enzyme. The catalytic site of the enzyme is in the P subunit or at the interface between the α and β subunits of the membrane extrinsic F1 sector. The proton pathway is formed from the a, b, and c subunits of the membrane intrinsic Fo sector. The γ, δ, and e subunits of F1 are required functionally and structurally to connect the catalytic subunits to the Fo sector. The mechanism of ATP hydrolysis can be studied using purified F1 (F1 — ATPase).