Amino acid sequence of the nucleotide binding region of chloroplast coupling factor 1

Abstract

The labeling of chloroplast coupling factor 1 by 3''-O-(4-benzoyl)benzoyl-ATP (BzATP) was studied. When the enzyme was incubated with .apprx. 10 .mu.M BzATP and 6 mM MgCl2 at pH 7.9 for .apprx. 20 min and passed through two Sephadex G-50 centrifuge columns, three BzATP molecules were bound per coupling factor molecule. Photolysis of radioactive enzyme-bound BzATP followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the BzATP bound primarily to the .beta.-polypeptide. If unbound BzATP was not removed by centrifuge columns prior to photolysis, significant labeling of the .alpha.-polypeptide also occurred. After photolysis, the BzATP-labeled enzyme was treated with trypsin, and two radioactive peptides were isolated by high-performance liquid chromatography on a C18 column. The two peptides were sequenced and found to correspond to amino acids 360-378 and 393-397 of the .beta.-polypeptide. For the sequence 360-378, two specific amino acids were found to be radioactive (Tyr-362 and Asp-369). This region of the polypeptide is highly conserved in several different species and probably corresponds to part of the nucleotide binding region of the catalytic site. In the case of amino acids 393-397, a very low level of radioactivity was found for all amino acids. The significance of this peptide for the binding of nucleotides to coupling factor 1 could not be established.