Crystallographic data of OAOR in complex with its two substrates reveals the str

Question

Crystallographic data of OAOR in complex with its two substrates reveals the structure of the enzyme's active site and Michaelis complex, shown below (the solid line indicates the surface of the protein). While the active site and oxaloacetate are both highly charged molecules, the crystallographic data indicates that a number of intermolecular forces, in addition to ionic contacts, are present in the the Michaelis complex. Clearly indicate these interactions in the figure below; note that some of these interactions do not occur with substrate and that a salt bridge does not preclude another type of interaction, such as an H-bond. The K_M of OAOR for NADH is approximately 1000-fold smaller the K_M of OAOR for NAD^+. Based on the structure of the active site, explain this phenomenon.

Explanation / Answer

Oxidation of NADH by decavanadate, a polymeric form vanadate with a cage-like structure, in presence of rat liver microsomes followed a biphasic pattern. An initial slow phase involved a small rate of oxygen uptake and reduction of 3 of the 10 vanadium atoms. This was followed by a second rapid phase in which the rates of NADH oxidation and oxygen uptake increased several-fold with a stoichiometry of NADH: O2 of 1:1. The burst of NADH oxidation and oxygen uptake which occurs in phosphate, but not in Tris buffer, was prevented by SOD, catalase, histidine, EDTA, MnCl2 and CuSO4, but not by the hydroxyl radical quenchers, ethanol, methanol, formate and mannitol. The burst reaction is of a novel type that requires the polymeric structure of decavanadate for reduction of vanadium which, in presence of traces of H2O2, provides a reactive intermediate that promotes transfer of electrons from NADH to oxygen.

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