Kinetic characterization of recombinant human cytosolic phosphoenolpyruvate carboxykinase with and without a His(10)-tag.

摘要

We report the first kinetic characterization of human liver cytosolic GTP-dependent phosphoenolpyruvate carboxykinase (GTP-PEPCK), which plays a major role in the development of type 2 diabetes in human. In this work two recombinant forms of the enzyme were studied. One form had a His(10)-tag and the other was His-tag-free, and with one exception, both exhibited similar kinetic properties. When Mn(2+) was used as the sole divalent cation, the His(10)-tagged enzyme, but not the His-tag-free enzyme, was increasingly inhibited at Mn(2+) concentrations greater than 0.7 mM. This inhibition did not pose any problem in kinetic analysis, for within the relevant Mn(2+) concentration range the His-tagged human PEPCK behaved almost identically to the tag-free enzyme. This property will bring simplicity and speed to purifying and studying multiple structural variants of this important enzyme. Apparent K(m) values of tag-free enzyme for phosphoenolpyruvate, GDP and bicarbonate were 450, 79 and 20,600 muM, respectively, while those for oxaloacetate and GTP were 4 and 23 muM, respectively, emphasizing the enzyme's gluconeogenic character. Bicarbonate (>100 mM) inhibited OAA-forming activity, which was a new observation with a GTP-PEPCK. The apparent K(m) for Mn(2+) in the PEP-forming direction was 30-fold lower than that for the OAA-forming direction. Mn(2+) and bicarbonate or CO(2) might regulate the enzyme in vivo.