Pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), an adenine nucleotide affinity analog, was found to bind in a saturable fashion to isolated α-subunit from Escherichia coli F1-ATPase with a stoichiometry of one mol/mol and a K(d) ~ 150 μM. The binding was shown to be specific by the following criteria: 1) ATP reduced the binding of PLO-AMP by 80%, and 2) PLP-AMP, like ATP, induced a conformational change which increased the mobility of α-subunit in nondenaturing polyacrylamide gel electrophoresis and rendered α-subunit resistant to mild trypsin proteolysis. A stable adduct was formed between isolated α-subunit and [3H] PLP-AMP after reduction with NaBH4. α-Subunit labeled to the extent of 0.4-0.7 mol/mol was digested with trypsin and subjected to high pressure liquid chromatography purification, yielding a single labeled peptide. Automated amino acid sequencing showed that residue α-Lys-201 was specifically labeled. The results suggest that Lys-201 occupies a position proximate to the phosphate groups of bound ATP in the α·ATP complex. PLP-AMP did not support repolymerization of isolated α-, β-, and γ-subunits, consistent with previous reports that subunit repolymerization in vitro is dependent upon the presence of nucleoside triphosphate. Further, PLP-AMP-labeled α-subunit could not be reconstituted with isolated β- and γ-subunits in the presence of ATP, showing that occupation of the α-subunit nucleotide site by PLP-AMP impairs normal subunit-subunit interaction.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - 1988|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology