Effects of Mn 2+ on the exchange reaction of phospho enolpyruvate carboxykinase in the presence of high concentrations of Mg 2+

Abstract

The mitochondrial phospho enolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32), purified from chick embryo liver, was synergistically activated by a combination of Mn 2+ and Mg 2+ in the oxaloacetate ↔ H 14CO 3 − exchange reaction. Increases in the Mg 2+ concentration caused decreases in the K 0.5 value of Mn 2+ in line with the earlier finding that the enzyme was markedly activated by low Mn 2+ (μM) plus high Mg 2+ (mM). In the presence of 2.5 mM Mg 2+, increases in the Mn 2+ level first enhanced the activity of phospho enolpyruvate carboxykinase, and then suppressed it to the maximal velocity shown in the presence of Mn 2+ alone. Kinetic studies showed that high Mn 2+ inhibited the activity of Mg 2+ noncompetitively, and those of GTP and oxaloacetate uncompetitivel. The inhibition constant for oxaloacetate ( K i′ = 550 μM) was lower than that of Mg 2+ ( K i = K i′ = 860 μM) or GTP ( K i′ = 1.6 mM), and was nearly equal to the apparent half-maximal inhibition concentration of Mn 2+. These results suggested that Mn 2+ can play two roles, of activating and suppressing phospho enolpyruvate carboxykinase activity in the presence of high Mg 2+.