Kinetic characterization of recombinant human cytosolic phosphoenolpyruvate carboxykinase with and without a His10-tag.
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 His10-tag and the other was His-tag-free, and with one exception, both exhibited similar kinetic properties. When Mn2+ was used as the sole divalent cation, the His10-tagged enzyme, but not the His-tag-free enzyme, was increasingly inhibited at Mn2+ concentrations greater than 0.7 mM. This inhibition did not pose any problem in kinetic analysis, for within the relevant Mn2+ 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 Km values of tag-free enzyme for phosphoenolpyruvate, GDP and bicarbonate were 450, 79 and 20,600 microM, respectively, while those for oxaloacetate and GTP were 4 and 23 microM, 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 Km for Mn2+ in the PEP-forming direction was 30-fold lower than that for the OAA-forming direction. Mn2+ and bicarbonate or CO2 might regulate the enzyme in vivo.
Case
Christopher L
CL
Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
Mukhopadhyay
Biswarup
B
D000818:Animals; D002413:Cations, Divalent; D003600:Cytosol / Q000201:enzymology*; D006168:Guinea Pigs; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics
D002413:Cations, Divalent; D011994:Recombinant Proteins; C033223:polyhistidine; D008345:Manganese; D006639:Histidine; D010729:Phosphoenolpyruvate Carboxykinase (GTP)