Pyridoxal 5'-diphospho-5'-adenosine binds at a single site on isolated alpha-subunit from Escherichia coli F1-ATPase and specifically reacts with lysine 201.
Pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), an adenine nucleotide affinity analog, was found to bind in a saturable fashion to isolated alpha-subunit from Escherichia coli F1-ATPase with a stoichiometry of one mol/mol and a Kd approximately 150 microM. The binding was shown to be specific by the following criteria: 1) ATP reduced the binding of PLP-AMP by 80%, and 2) PLP-AMP, like ATP, induced a conformational change which increased the mobility of alpha-subunit in nondenaturing polyacrylamide gel electrophoresis and rendered alpha-subunit resistant to mild trypsin proteolysis. A stable adduct was formed between isolated alpha-subunit and [3H] PLP-AMP after reduction with NaBH4. alpha-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 alpha-Lys-201 was specifically labeled. The results suggest that Lys-201 occupies a position proximate to the phosphate groups of bound ATP in the alpha.ATP complex. PLP-AMP did not support repolymerization of isolated alpha-, beta-, and gamma-subunits, consistent with previous reports that subunit repolymerization in vitro is dependent upon the presence of nucleoside triphosphate. Further, PLP-AMP-labeled alpha-subunit could not be reconstituted with isolated beta- and gamma-subunits in the presence of ATP, showing that occupation of the alpha-subunit nucleotide site by PLP-AMP impairs normal subunit-subunit interaction.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives*; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D001894:Borohydrides; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D008239:Lysine / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011487:Protein Conformation / Q000187:drug effects; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D001894:Borohydrides; D046911:Macromolecular Substances; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; C025364:sodium borohydride; D000255:Adenosine Triphosphate; D014357:Trypsin; D006180:Proton-Translocating ATPases; C048236:pyridoxal 5'-diphospho-5'-adenosine; D008239:Lysine