Studies of enzyme-ligand complexes using dynamic fluorescence anisotropy. II. The coenzyme-binding site of malate dehydrogenase.
The coenzyme-binding site in mitochondrial malate dehydrogenase from pig heart was studied using dynamic fluorescence anisotropy decay. The dynamics of the fluorescent ligands NADH and 6-cyano-7-hydroxy-4,8-dimethylcoumarin were used to detect conformational changes at the dihydronicotinamide-and at the adenosine-binding sites, respectively. Addition of the natural substrate L-malate to the complex from enzyme and NADH does not influence the complete immobilization of the dihydronicotinamide group, whereas the stereoisomer D-malate and the substrate-analogue hydroxymalonate form ternary complexes with highly mobile dihydronicotinamide. The dynamics of the fluorescent adenosine-analogue are not influenced by formation of complexes with substrate and substrate-analogues. Thus the conformational changes at the dihydronicotinamide-binding site remain local and are not transmitted to the adenosine-binding site.
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003374:Coumarins; D005454:Fluorescence Polarization; D006801:Humans; D008291:Malate Dehydrogenase; D008929:Mitochondria, Heart / Q000201:enzymology; D009243:NAD / Q000378:metabolism; D013552:Swine
D003374:Coumarins; D009243:NAD; C048700:6-cyano-7-hydroxy-4,8-dimethylcoumarin; D008291:Malate Dehydrogenase; D000241:Adenosine