Studies of Glutamate Dehydrogenase

Glyoxal inhibits the catalytic reaction of glutamate dehydrogenase competitively with respect to 2‐oxoglutarate, whereas the hydrodynamic and allosteric properties remain largely unchanged upon treatment with glyoxal. The reaction is completely reversible, but the Schiff base intermediate can be trapped by reduction with NaBH 4 . Determination of the free amino groups indicates that glyoxal reacts with one amino group per polypeptide chain. From the analysis of K m and V in the range of pH 7.8 to pH 9.2 it is concluded that this amino group has a p K ‐value of 8.2 and is responsible in its protonated state for the binding of the substrate 2‐oxoglutarate. 5‐Diazo‐1 H ‐tetrazole reacts with glutamate dehydrogenase predominantly by modifying amino groups. Depending on the reaction conditions 5‐diazo‐1 H ‐tetrazole first modifies that amino group which is responsible for the binding of the substrate 2‐oxoglutarate and secondly the amino group responsible for the binding of the dihydronicotinamide part of NADH. If the modification is performed in the presence of the substrate 2‐oxoglutarate the amino group which binds the substrate is protected. The modification of the enzyme with 5‐diazo‐1 H ‐tetrazole also conserves to a large extent the ability of the oligomer to associate and its allosteric properties.