ATP turnover by the fatty acid reductase complex of Photobacterium phosphoreum

A sensitive reverse-phase high pressure liquid chromatographic assay for formation of AMP coupled with analysis of aldehyde production has been used to characterize the properties of the fatty acid reductase complex of Photobacterium phosphoreum. The enzyme complex, which consists of three different polypeptides (34 000, 50 000, and 58 000), has a high affinity for ATP (Km = 20 nM) and shows highest specificity with C14 fatty acids. Activation of the fatty acid is efficiently coupled to the reduction step showing a stoichiometry of one molecule of fatty acid reduced to aldehyde and one molecule of NADPH oxidized for every molecule of ATP converted to AMP. Reconstituted fatty acid reductase (50 000 and 58 000) shows an ATP hydrolase activity that is independent of NADPH with the maximum amount of AMP formed limited by the amount of fatty acid in the assay, consistent with acyl-protein turnover experiments and the channeling of fatty acids to form acyl thioesters (−NADPH) or aldehyde (+NADPH). Addition of t...