Coenzyme Binding to L-α-Glycerophosphate Dehydrogenase

The binding of NADH to rabbit muscle l-α-glycerophosphate dehydrogenase is accompanied by a considerable enhancement of the fluorescence of the reduced coenzyme which was used to study the properties of this binding process. Fluorescence titration of the enzyme with NADH indicated the binding of 2 moles of NADH per mole of enzyme (78,000 g). The dissociation constant for the NADH-enzyme complex was determined to be 1.8 x 10-6 m. Adenylic acid, adenosine diphosphate, and adenosine diphosphoribose were shown to be NADH-competitive inhibitors in the l-α-glycerophosphate dehydrogenase-catalyzed reduction of dihydroxyacetone phosphate. A competitive relationship between the binding of adenylic acid and NADH was also shown with fluorescence enhancement techniques. Peptide-mapping studies of trypsin digests of l-α-glycerophosphate dehydrogenase revealed the presence in this enzyme of two identical subunits of 39,000 molecular weight.