ROLE OF THE DIVALENT METAL-ION IN THE NAD-MALIC ENZYME REACTION - AN ESEEM DETERMINATION OF THE GROUND-STATE CONFORMATION OF MALATE IN THE E-MN-MALATE COMPLEX

The conformation of L-malate bound at the active site of Ascaris suum malic enzyme has been investigated by electron spin echo envelope modu lation spectroscopy. Dipolar interactions between Mn2+ bound to the en zyme active site and deuterium specifically placed at the 2-position, the 3R-position, and the 3S-position of L-malate were observed. The in tensities of these interactions are related to the distance between ea ch deuterium and Mn2+ Several models of possible Mn-malate complexes w ere constructed using molecular graphics techniques, and conformationa l searches were conducted to identify conformers of malate that meet t he distance criteria defined by the spectroscopic measurements. These searches suggest that L-malate binds to the enzyme active site in the trans conformation, which would be expected to be the most stable conf ormer in solution, not in the gauche conformer, which would be more si milar to the conformation required for oxidative decarboxylation of ox alacetate formed from L-malate at the active site of the enzyme.