Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labelling: binding characteristics and amino acid sequences

5-Lipoxygenase (5LO) catalyses the first two steps in the biosynthesis of l eukotrienes, which are inflammatory mediators derived from arachidonic acid . 5LO activity is stimulated by ATP; however, a consensus ATP-binding site or nucleotide-binding site has not been found in its protein sequence. In t he present study, affinity and photoaffinity labelling of 5LO with 5'- p-fl uorosulphonylbenzoyladenosine (FSBA) and 2-azido-ATP showed that 5LO bound to the ATP analogues quantitatively and specifically and that the incorpora tion of either analogue inhibited ATP stimulation of 5LO activity. The stoi chiometry of the labelling was 1.4 mol of FSBA/mol of 5LO (of which ATP com peted with 1 mol/mol) or 0.94 mol of 2-azido-ATP/mol of 5LO (of which ATP c ompeted with 0.77 mol/mol). Labelling with FSBA prevented further labelling with 2-azido-ATP, indicating that the same binding site was occupied by bo th analogues. Other nucleotides (ADP, AMP, GTP, CTP and UTP) also competed with 2-azido-ATP labelling, suggesting that the site was a general nucleoti de-binding site rather than a strict ATP-binding site. Ca2+, which also sti mulates 5LO activity, had no effect on the labelling of the nucleotide-bind ing site. Digestion with trypsin and peptide sequencing showed that two fra gments of 5LO were labelled by 2-azido-ATP. These fragments correspond to r esidues 73-83 (KYWLNDDWYLK, in single-letter amino acid code) and 193-209 ( FMHMFQSSWNDFADFEK) in the 5LO sequence. Trp-75 and Trp-201 in these peptide s were modified by the labelling, suggesting that they were immediately adj acent to the C-2 position of the adenine ring of ATP. Given the stoichiomet ry of the labelling, the two peptide sequences of 5LO were probably near ea ch other in the enzyme's tertiary structure, composing or surrounding the A TP-binding site of 5LO.