A GTP-dependent Vertebrate-type Phosphoenolpyruvate Carboxykinase from Mycobacterium smegmatis

This is the first report on a bacterial verterbrate-type GTP-dependent phosphoenolpyruvate carboxykinase (PCK). The pck gene of Mycobacterium smegmatis was cloned. The recombinant PCK was overexpressed inEscherichia coli in a soluble form and with high activity. The purified enzyme was found to be monomeric (72 kDa), thermophilic (optimum temperature, 70 °C), very stable upon storage at 4 °C, stimulated by thiol-containing reducing agents, and inhibited by oxalate and by α-ketoglutarate. The requirement for a divalent cation for activity was fulfilled best by Mn2+ and Co2+ and poorly by Mg2+. At 37 °C, the highest Vm value (32.5 units/mg) was recorded with Mn2+ and in the presence of 37 mmdithiothreitol (DTT). The presence of Mg2+ (2 mm) greatly lowered the apparent Kmvalues for Mn2+ (by 144-fold in the presence of DTT and by 9.4-fold in the absence of DTT) and Co2+ (by 230-fold). In the absence of DTT but in the presence of Mg2+ (2 mm) as the co-divalent cation, Co2+ was 21-fold more efficient than Mn2+. For producing oxaloacetate, the enzyme utilized both GDP and IDP; ADP served very poorly. The apparent Km values for phosphoenolpyruvate, GDP, and bicarbonate were >100, 66, and 8300 μm, respectively, whereas those for GTP and oxaloacetate (for the phosphoenolpyruvate formation activity) were 13 and 12 μm, respectively. Thus, this enzyme preferred the gluconeogenesis/glycerogenesis direction. This property fits the suggestion that in M. smegmatis, pyruvate carboxylase is not anaplerotic but rather gluconeogenic (Mukhopadhyay, B., and Purwantini, E. (2000) Biochim. Biophys. Acta. 1475, 191–206). Both in primary structure and kinetic properties, the mycobacterial PCK was very similar to its vertebrate-liver counterparts and thus could serve as a model for these enzymes; examples for several immediate targets are presented.