STUDY OF PHOSPHOGLYCERATE KINASE IN HUMAN ERYTHROCYTES .2. KINETIC-PROPERTIES

Kinetic studies on phosphoglycerate kinase (EC 2.7.2.3) were performed in the forward reaction leading from 1,3-diphosphoglycerate to 3-phosphoglycerate. Substrate activation was observed at fixed levels of ADP or Mg2+ and varying concentrations of 1,3-diphosphoglycerate. A biphasic curve was obtained in both linear and double reciprocal plots demonstrating 2 Km values (Km1 1.9 .cntdot. 10-6 and Km2 9.8 .cntdot. 10-6 M). Michaelis-Menten-type kinetics were observed in both the linear and double reciprocal plots at fixed levels of 1,3-diphosphoglycerate or ADP and varying concentrations of Mg2+. Apparent Michaelis-Menten kinetics were observed in linear plots when conditions of fixed concentrations of 1,3-diphosphoglycerate or Mg2+ were maintained with varying concentrations of ADP. The double-reciprocal plots demonstrated biphasic curves with 2 Km values (Km1 1.7 .cntdot. 10-5 and Km2 1.0 .cntdot. 10-4 M). Apparent negative cooperativity was observed with respect to 1,3-diphosphoglycerate and ADP. Phosphoglycerate kinase activity was inhibited by AMP and 2,3-diphosphoglycerate. Substrate activation by 1,3-diphosphoglycerate was maintained in the presence of AMP or 2,3-diphosphoglycerate but at a reduced level of enzyme activity. AMP inhibited enzyme activity non-competitively with respect to 1,3-diphosphoglycerate, ADP and Mg2+. 2,3-Diphosphoglycerate inhibits phosphoglycerate kinase activity with respect to 1,3-diphosphoglycerate, ADP and Mg2+. 2,3-Diphosphoglycerate inhibits phosphoglycerate kinase activity non-competitively with respect to 1,3-diphosphoglycerate.