Purification, characterization, and substrate and inhibitor structure-activity studies of rat liver FAD-AMP lyase (cyclizing): Preference for FAD andspecificity for splitting ribonucleoside diphosphate-X into ribonucleotideand a five-atom cyclic phosphodiester of X, either a monocyclic compound or a cis-bicyclic phosphodiester-pyranose fusion

An enzyme with FAD-AMP lyase (cyclizing) activity, splitting FAD to AMP and riboflavin 4',5'-phosphate (cFMN), was recently identified [Fraiz, F., et al. (1998) Biochem. J. 330, 881-888]. Now, it has been purified to apparent homogeneity from a rat liver supernatant, by a procedure that includes aff inity for ADP-agarose (adsorption required the activating cation Mn2+ and d esorption required its removal), to a final activity of 2.2 units/mg after a 240-fold purification with a 15% yield. By SDS-PAGE, only one protein ban d was observed (M-r = 59 000). The correspondence between protein and enzym e activity was demonstrated by renaturation after SDS-PAGE, by gradient ult racentrifugation followed by analytical SDS-PAGE, and by native PAGE with v isualization of enzyme activity by fluorescence. A native M-r of 100 000 (u ltracentrifugation) or 140 000 (gel filtration) indicated that FAD-AMP lyas e could be a dimer. The enzyme required millimolar concentrations of Mn2+ o r Col, exhibited different optimum pH values with these cations (pH 8.5 or 7.3,. respectively), and was strongly inhibited by ADP or ATP, but not by d ADP, dATP, or the reaction products AMP and cFMN. A specificity study was c onducted with 35 compounds related to FAD, mostly nucleoside diphosphate-X (NDP-X) derivatives. Besides FAD, the enzyme split 11 of these compounds wi th the pattern NDP-X --> NMP + P=X. Structure-activity correlations of subs trates, nonsubstrates, and inhibitors, and the comparison of the enzymic re activities of NDP-X compounds with their susceptibilities to metal-dependen t chemical degradation, pinpointed the following specificity pattern. FAD-A MP lyase splits ribonucleoside diphosphate-X compounds in which X is an acy clic or cyclic monosaccharide or derivative bearing an X-OH group that is a ble to attack internally the proximal phosphorus with the geometry necessar y to form a P=X product, either a five-atom monocyclic phosphodiester or a cis-bicyclic phosphodiester-pyranose fusion. For instance, NDP-glucose and GDP-alpha -L-fucose were substrates, but dTDP-glucose, NDP-mannose, and GDP -beta -L-fucose were not. Judging from k(cat)/K-m ratios, we found the best substrate to be FAD, followed closely by ADP-glucose (k(cat)/K-m only 2-fo ld lower, but not a physiological compound in mammals), whereas other subst rates exhibited 50-500-fold lower k(cat)/K-m values. However, there was no evidence for specific flavin recognition. Instead, what seems to be recogni zed is the NDP moiety of NDP-X, with a strong preference for ADP-X. Splitti ng would then depend on the presence of an adequate X-OH group. The possibi lity that, besides FAD, there could be in mammals other ADP-X substrates of FAD-AMP lyase is discussed, with emphasis placed on soiree ADP-ribose deri vatives.