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 in Escherichia coli in a soluble form and with high activity. The purified enzyme was found to be monomeric (72 kDa), thermophilic (optimum temperature, 70 degreesC), very stable upon storage at 4 degreesC, stimulated by thiol-containing reducing agents, and inhibited by oxalate and by cu-ketoglutarate. The requirement for a divalent cation for activity was fulfilled best by Mn2+ and Co2+ and poorly by Mg2+. At 37 degreesC, the highest V-m value (32.5 units/mg) was recorded with Mn2+ and in the presence of 37 mM dithiothreitol (DTT), The presence of Mg2+ (2 mM) greatly lowered the apparent K-m, values 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 K-m values for phosphoenolpyruvate, GDP, and bicarbonate were >100, 66, and 8300 muM, respectively, whereas those for GTP and oxaloacetate (for the phosphoenolpyruvate formation activity) were 13 and 12 muM, 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 PCR 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.