A short sequence motif rich in glycine residues, Gly-X-X-X-X-Gly-Lys-Thr/Ser, has been found in many nucleotide-binding proteins including the β subunit of Escherichia coli H+-ATPaSe (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residues 149-156). The following mutations were introduced in this region of the cloned E. coli unc operon carried by a plasmid pBWU1: Ala-151 → Pro or Val; insertion of a Gly residue between Lys-155 and Thr-156; and replacement of the region by the corresponding sequence of adenylate kinase (Gly-Gly-Pro-Gly-Ser-Gly-Lys-Gly-Thr) or p21 ras protein (ras) (Gly-Ala-Gly-Gly-Val-Gly-Lys-Ser). All F0F1 subunits were synthesized in the deletion strain of the unc operon-dependent on pBWU1 with mutations, and essentially the same amounts of H+-ATPaSe with these mutant β subunits were found in membranes. The adenylate kinase and Gly insertion mutants showed no oxidative phosphorylation or ATPase activity, whereas the Pro-151 mutants had higher ATPase activity than the wild-type, and the Val-151 and ras mutants had significant activity. It is striking that the enzyme with the ras mutation (differing in three amino acids from the β sequence) had about half the membrane ATPase activity of the wild-type. These results together with the simulated three-dimensional structures of the wild-type and mutant sequences suggest that in mutant β subunits with no ATPase activity projection of Thr-156 residues was opposite to that in the wild-type, and that the size and direction of projection of residue 151 are important for the enzyme activity.
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - Dec 5 1990|
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