Abstract
Phosphoglycerate kinase (ATP: 3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) from Escherichia coli AHU1520 was purified until electrophoretically homogeneous by a single procedure, using a linear gradient elution of 2, 3-bisphosphoglycerate between 0 and 0.5 mM on a blue dextran-Sepharose 4B column chromatography. The phosphoglycerate kinase was inhibited by blue dextran competitively with respect to 3-phosphoglycerate, and noncompetitively with respect to
ATP. The inhibition of the kinase by Cibacron Blue was competitive with respect to 3-phosphoglycerate at both saturated and unsaturated levels of ATP; the inhibition by the dye toward ATP was shifted from a mixed-type at a saturated level of 3-phosphoglycerate to competitive-type at an unsaturated level. Further inhibition studies implied that two molecules of the dye were bound per molecule of phosphoglycerate kinase with an unsaturated level of ATP used as a fixed substrate. The shape of the difference spectrum of Cibacron Blue in the presence of the kinase was very similar to that of a difference spectrum generated upon addition of KCl, but was markedly different from the shape of the spectrum produced by the addition of glycerol.
