A GTP-dependent vertebrate-type phosphoenolpyruvate carboxykinase from Mycobacterium smegmatis

This is the first report on a bacterial verterbrate-type GTP-dependent phos phoenolpyruvate carboxykinase (PCK). The pck gene of Mycobacterium smegmati s 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 s table upon storage at 4 degreesC, stimulated by thiol-containing reducing a gents, and inhibited by oxalate and by cu-ketoglutarate. The requirement fo r a divalent cation for activity was fulfilled best by Mn2+ and Co2+ and po orly by Mg2+. At 37 degreesC, the highest V-m value (32.5 units/mg) was rec orded with Mn2+ and in the presence of 37 mM dithiothreitol (DTT), The pres ence of Mg2+ (2 mM) greatly lowered the apparent K-m, values for Mn2+ (by 1 44-fold in the presence of DTT and by 9.4-fold in the absence of DTT) and C o2+ (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 ve ry poorly. The apparent K-m values for phosphoenolpyruvate, GDP, and bicarb onate were >100, 66, and 8300 muM, respectively, whereas those for GTP and oxaloacetate (for the phosphoenolpyruvate formation activity) were 13 and 1 2 muM, respectively. Thus, this enzyme preferred the gluconeogenesis/glycer ogenesis direction, This property fits the suggestion that in M. smegmatis, pyruvate carboxylase is not anaplerotic but rather gluconeogenic (Mukhopad hyay, B., and Purwantini, E. (2000) Biochim. Biophys. Acta. 1475, 191-206). Both in primary structure and kinetic properties, the mycobacterial PCR wa s very similar to its vertebrate-liver counterparts and thus could serve as a model for these enzymes; examples for several immediate targets are pres ented.