Kinetic Studies of Dogfish Liver Glutamate Dehydrogenase with Diphosphopyridine Nucleotide and the Effect of Added Salts*

Kinetic studies have been performed with crystalline dogfish liver glutamate dehydrogenase with the diphosphopyridine nucleotides as cofactors. The kinetic constants for the various substrates have been determined. In its sensitivity to guanosine S-triphosphate, adenosine S&phosphate, and excess reduced diphosphopyridine nucleotide the enzyme is similar to that derived from other vertebrates. The extent of inhibition of the initial rate of the aminating reaction by excess reduced diphosphopyridine nucleotide was reduced by the presence of increased ammonium chloride in the reaction mixture. The effect is attributable to the chloride ion. A number of anions have been tested, and the order of their effectiveness in preventing inhibition by excess reduced coenzyme followed the Hofmeister series: C104> SCN> I> Br> Cl> F-. Peak effectiveness was observed at comparatively low concentrations of salts, varying from 0.045 M C104to 0.25 M Cl-. Further increase in salt concentration caused a progressive decrease in the rate of reaction. Concentrations of salts that were effective in preventing inhibition by excess reduced diphosphopyridine nucleotide were observed to depress activity at noninhibitory levels of coenzyme. Added salt does not affect the calculated maximum velocity of the reaction but does increase the apparent K,,, for the diphosphopyridine nucleotide and effectively prevents inhibition at high concentrations of the cofactor. The presence of added salt minimized the regulatory influence of the nucleotides guanosine 5’4riphosphate and adenosine S-diphosphate.