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J. Biol. Chem., Vol. 276, Issue 19, 16137-16145, May 11, 2001

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

Biswarup Mukhopadhyay, Edward M. Concar, and Ralph S. Wolfe

From the Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

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 °C), very stable upon storage at 4 °C, stimulated by thiol-containing reducing agents, and inhibited by oxalate and by -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 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 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF332191.

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