The Mechanism of End Product Inhibition of Serine Biosynthesis

Phosphoglycerate dehydrogenase was purified from extracts of Escherichia coli by conventional methods and the enzyme was crystallized from ammonium sulfate solution. The crystalline preparation appeared homogenous in the ultracentrifuge and upon zone electrophoresis. The reduction of hydroxypyruvate-P proceeded faster than the oxidation of phosphoglycerate (PGA) and the latter reaction was inhibited by DPNH. The equilibrium value measured in the direction of PGA oxidation was ‘7 x lo-l1 M at pH 7.5 and 25’. L Serine in the range of 1 to 10 pM inhibited the PGA dehydrogenase-catalyzed oxidation of PGA and reduction of hydroxypyruvate-P. Maximum inhibition was about 95%. To get inhibition with other amino acids concentrations of 1 mu and higher were required. The dose-response curves for serine were sigmoid-shaped and when converted to Hill plots gave slopes that approached 2. L-Serine was a noncompetitive inhibitor with respect to hydroxypyruvate-P, an uncompetitive inhibitor with respect to PGA and DPNH, and gave “mixed” type inhibition with respect to DPN. The substrates gave regular MichaelisMenton kinetics. Analogues of DPNH functioned as coenzymes but TPNH had only 7.5% the activity of DPNH.