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J. Biol. Chem., Vol. 276, Issue 19, 16137-16145, May 11, 2001
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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 The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF332191.
A GTP-dependent Vertebrate-type Phosphoenolpyruvate
Carboxykinase from Mycobacterium smegmatis*
,
-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 mM
dithiothreitol (DTT). The presence of Mg2+ (2 mM) greatly lowered the apparent Km
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 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.
*
This work was supported by Department of Energy Grant
DE-FG02-87ER13651 and National Institutes of Health Grant GM 51334.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom all correspondence should be addressed: Dept. of
Microbiology, B103 Chemical and Life Sciences Laboratory, University of
Illinois at Urbana-Champaign, 601 S. Goodwin Ave., Urbana, IL 61801. Tel.: 217 333 1397; Fax: 217 244 6697; E-mail:
biswarup@life.uiuc.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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