Reaction between Reduced Diphosphopyridine Nucleotide and Glutamate Oxalacetate Transaminases

Abstract

Abstract DPNH can be bound to pig heart cytoplasmic and bovine liver mitochondrial glutamate oxalacetate transaminases. The physiological function of this binding is not known, but DPNH does protect the mitochondrial enzyme from inactivation. Not only can DPNH be bound, but in the presence of ammonium ions it can be oxidized to DPN. This reaction is inhibited by phenylhydrazine. Consequently, it is believed that in this reaction a keto group which is tightly bound to the transaminase is reductively aminated. The product of the reaction other than DPN has not yet been identified. If DPNH with tritium on the B-side is incubated, then tritium is transferred from DPNH to water. While some pyridoxamine phosphate can be produced, it is produced in considerably lesser amounts than DPN. These reactions are apparently not the result of an artifact induced during the purification of the enzymes; they are quite slow and apparently take place independent of reactions between glutamate dehydrogenase and transaminase-bound pyridoxal or pyridoxamine phosphate. Additional evidence is presented which suggests that glutamate dehydrogenase can form an enzyme-enzyme complex with the transaminase, and in the presence of DPNH (or TPNH) plus ammonium ions, glutamate dehydrogenase reacts with transaminase-bound pyridoxal phosphate to produce equal amounts of DPN (or TPN) and pyridoxamine phosphate. In the presence of DPN (or TPN), glutamate dehydrogenase reacts with transaminase-bound pyridoxamine phosphate to produce equal amounts of DPNH (or TPNH) plus pyridoxal phosphate. In these reactions glutamate dehydrogenase is apparently reacting directly with pyridoxal or pyridoxamine phosphate. That is, these reactions are apparently not mediated by some keto or amino acid. Glutamate dehydrogenase must have direct physical contact with the transaminase for there to be interaction between the two enzymes. In the enzyme-enzyme complex, glutamate dehydrogenase is the actual catalyst and transaminase-bound pyridoxal or pyridoxamine phosphate is the substrate.