The Mechanism of End Product Inhibition of Serine Biosynthesis

The absorption spectrum of crystalline phosphoglycerate dehydrogenase had a peak at 276 mµ and a broad shoulder at 320 to 340 mµ. When activated in the region of 285 mµ the enzyme had a fluorescence emission spectrum with peaks in the region of 330 and 430 mµ. The 430 mµ peak was bleached when hydroxypyruvate phosphate was added. This result, plus the fluorescence activation spectrum, shows that the 430 mµ peak results from enzyme-bound DPNH. Addition of a solution of DPNH to the enzyme enhanced the 430 mµ peak and quenched the protein fluorescence at 330 mµ. The physiologically important inhibitor serine reduced the intensity of the 430 mµ peak. On the basis of changes in the fluorescence of the enzyme that accompany ligand binding, titrations were carried out with hydroxypyruvate phosphate, 3-phosphoglycerate, DPN, DPNH, analogues of DPNH, and serine. Dissociation constants were obtained and two binding sites per enzyme molecule were shown for hydroxypyruvate phosphate, DPNH, and serine. The titration curve for serine was sigmoidal, indicating interaction between the binding sites.