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 degrees C), very stable upon storage at 4 degrees C, stimulated by thiol-containing reducing agents, and inhibited by oxalate and by alpha-ketoglutarate. The requirement for a divalent cation for activity was fulfilled best by Mn(2+) and Co(2+) and poorly by Mg(2+). At 37 degrees C, the highest V(m) value (32.5 units/mg) was recorded with Mn(2+) and in the presence of 37 mm dithiothreitol (DTT). The presence of Mg(2+) (2 mm) greatly lowered the apparent K(m) values for Mn(2+) (by 144-fold in the presence of DTT and by 9.4-fold in the absence of DTT) and Co(2+) (by 230-fold). In the absence of DTT but in the presence of Mg(2+) (2 mm) as the co-divalent cation, Co(2+) was 21-fold more efficient than Mn(2+). 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 micrometer, respectively, whereas those for GTP and oxaloacetate (for the phosphoenolpyruvate formation activity) were 13 and 12 microm, 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.
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA. biswarup@life.uiuc.edu
B
B
Mukhopadhyay
E M
EM
Concar
R S
RS
Wolfe
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002712:Chlorides / Q000494:pharmacology; D003001:Cloning, Molecular; D004229:Dithiothreitol / Q000494:pharmacology; D004795:Enzyme Stability; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D017895:Manganese Compounds / Q000494:pharmacology; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014714:Vertebrates
D002712:Chlorides; D017895:Manganese Compounds; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D006160:Guanosine Triphosphate; D010729:Phosphoenolpyruvate Carboxykinase (GTP); C025340:manganese chloride; D004229:Dithiothreitol