PCKG_MYCSM
ID PCKG_MYCSM Reviewed; 605 AA.
AC Q9AGJ6;
DT 26-SEP-2001, integrated into UniProtKB/Swiss-Prot.
DT 01-JUN-2001, sequence version 1.
DT 03-AUG-2022, entry version 93.
DE RecName: Full=Phosphoenolpyruvate carboxykinase [GTP] {ECO:0000255|HAMAP-Rule:MF_00452, ECO:0000303|PubMed:11278451};
DE Short=PEP carboxykinase {ECO:0000255|HAMAP-Rule:MF_00452, ECO:0000303|PubMed:11278451};
DE Short=PEPCK {ECO:0000255|HAMAP-Rule:MF_00452, ECO:0000303|PubMed:11278451};
DE EC=4.1.1.32 {ECO:0000255|HAMAP-Rule:MF_00452, ECO:0000269|PubMed:11278451, ECO:0000269|PubMed:17015450};
DE AltName: Full=GTP-dependent phosphoenolpyruvate carboxykinase {ECO:0000303|PubMed:17015450};
DE Short=GTP-PEPCK {ECO:0000303|PubMed:17015450};
GN Name=pckG; Synonyms=pck;
OS Mycolicibacterium smegmatis (Mycobacterium smegmatis).
OC Bacteria; Actinobacteria; Corynebacteriales; Mycobacteriaceae;
OC Mycolicibacterium.
OX NCBI_TaxID=1772;
RN [1]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA], PROTEIN SEQUENCE OF 1-13, FUNCTION,
RP CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, ACTIVITY REGULATION,
RP COFACTOR, MASS SPECTROMETRY, SUBUNIT, AND SUBSTRATE SPECIFICITY.
RX PubMed=11278451; DOI=10.1074/jbc.m008960200;
RA Mukhopadhyay B., Concar E.M., Wolfe R.S.;
RT "A GTP-dependent vertebrate-type phosphoenolpyruvate carboxykinase from
RT Mycobacterium smegmatis.";
RL J. Biol. Chem. 276:16137-16145(2001).
RN [2]
RP DISRUPTION PHENOTYPE.
RX PubMed=12855734; DOI=10.1099/mic.0.26234-0;
RA Liu K., Yu J., Russell D.G.;
RT "pckA-deficient Mycobacterium bovis BCG shows attenuated virulence in mice
RT and in macrophages.";
RL Microbiology 149:1829-1835(2003).
RN [3]
RP FUNCTION, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, MUTAGENESIS OF
RP ASP-75; ASP-78 AND GLU-83, AND COFACTOR.
RX PubMed=17015450; DOI=10.1074/jbc.m602591200;
RA Case C.L., Concar E.M., Boswell K.L., Mukhopadhyay B.;
RT "Roles of Asp75, Asp78, and Glu83 of GTP-dependent phosphoenolpyruvate
RT carboxykinase from Mycobacterium smegmatis.";
RL J. Biol. Chem. 281:39262-39272(2006).
CC -!- FUNCTION: Involved in the gluconeogenesis, in growth on fatty acids and
CC is important for initiation of infection in the macrophages. Catalyzes
CC the GTP-dependent conversion of oxaloacetate (OAA) to
CC phosphoenolpyruvate (PEP), the rate-limiting step in the metabolic
CC pathway that produces glucose from lactate and other precursors derived
CC from the citric acid cycle. It can also use IDP or ADP, but with a
CC lower activity. {ECO:0000269|PubMed:11278451,
CC ECO:0000269|PubMed:17015450}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=GTP + oxaloacetate = CO2 + GDP + phosphoenolpyruvate;
CC Xref=Rhea:RHEA:10388, ChEBI:CHEBI:16452, ChEBI:CHEBI:16526,
CC ChEBI:CHEBI:37565, ChEBI:CHEBI:58189, ChEBI:CHEBI:58702; EC=4.1.1.32;
CC Evidence={ECO:0000269|PubMed:11278451, ECO:0000269|PubMed:17015450};
CC -!- COFACTOR:
CC Name=Mn(2+); Xref=ChEBI:CHEBI:29035;
CC Evidence={ECO:0000269|PubMed:11278451, ECO:0000269|PubMed:17015450};
CC Note=Binds 1 Mn(2+) ion per subunit. Can also use Co(2+) ion.
CC {ECO:0000269|PubMed:11278451, ECO:0000269|PubMed:17015450};
CC -!- ACTIVITY REGULATION: Inhibited by oxalate and by alpha-ketoglutarate.
CC In vitro, the enzyme is stimulated by reducing agents such as
CC dithiothreitol (DTT), reduced glutathione and 2-mercaptoethanol.
CC {ECO:0000269|PubMed:11278451}.
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Kinetic parameters:
CC KM=0.67 uM for OAA (PEP forming activity at pH 7.2 and 37 degrees
CC Celsius) {ECO:0000269|PubMed:17015450};
CC KM=3.3 uM for manganese (OAA forming activity at pH 7.2 and 37
CC degrees Celsius) {ECO:0000269|PubMed:17015450};
CC KM=69 uM for GDP (OAA forming activity at pH 7.2 and 37 degrees
CC Celsius) {ECO:0000269|PubMed:17015450};
CC KM=132 uM for manganese (OAA forming activity without DTT at pH 7.2
CC and 37 degrees Celsius) {ECO:0000269|PubMed:11278451};
CC KM=208 uM for cobalt (OAA forming activity without DTT at pH 7.2 and
CC 37 degrees Celsius) {ECO:0000269|PubMed:11278451};
CC KM=471 uM for PEP (OAA forming activity at pH 7.2 and 37 degrees
CC Celsius) {ECO:0000269|PubMed:17015450};
CC Vmax=8.6 umol/min/mg enzyme with manganese as substrate (OAA forming
CC activity without DTT at pH 7.2 and 37 degrees Celsius)
CC {ECO:0000269|PubMed:17015450};
CC Vmax=9.5 umol/min/mg enzyme with cobalt as substrate (OAA forming
CC activity without DTT at pH 7.2 and 37 degrees Celsius)
CC {ECO:0000269|PubMed:17015450};
CC Vmax=21 umol/min/mg enzyme with OAA as substrate (PEP forming
CC activity at pH 7.2 and 37 degrees Celsius)
CC {ECO:0000269|PubMed:17015450};
CC Vmax=32 umol/min/mg enzyme with manganese as substrate (OAA forming
CC activity at pH 7.2 and 37 degrees Celsius)
CC {ECO:0000269|PubMed:17015450};
CC Vmax=33.6 umol/min/mg enzyme with GDP as substrate (OAA forming
CC activity at pH 7.2 and 37 degrees Celsius)
CC {ECO:0000269|PubMed:17015450};
CC Vmax=38.2 umol/min/mg enzyme with PEP as substrate (OAA forming
CC activity at pH 7.2 and 37 degrees Celsius)
CC {ECO:0000269|PubMed:17015450};
CC pH dependence:
CC Optimum pH is between 7 and 7.4. {ECO:0000269|PubMed:11278451};
CC Temperature dependence:
CC Optimum temperature is 70 degrees Celsius.
CC {ECO:0000269|PubMed:11278451};
CC -!- PATHWAY: Carbohydrate biosynthesis; gluconeogenesis. {ECO:0000305}.
CC -!- SUBUNIT: Monomer. {ECO:0000269|PubMed:11278451}.
CC -!- SUBCELLULAR LOCATION: Cytoplasm {ECO:0000305}.
CC -!- MASS SPECTROMETRY: Mass=71209; Method=MALDI;
CC Evidence={ECO:0000269|PubMed:11278451};
CC -!- DISRUPTION PHENOTYPE: Cells lacking this gene are inhibited when they
CC grow on acetate and palmitate. {ECO:0000269|PubMed:12855734}.
CC -!- SIMILARITY: Belongs to the phosphoenolpyruvate carboxykinase [GTP]
CC family. {ECO:0000305}.
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DR EMBL; AF332191; AAK28534.1; -; Genomic_DNA.
DR AlphaFoldDB; Q9AGJ6; -.
DR SMR; Q9AGJ6; -.
DR BRENDA; 4.1.1.32; 3512.
DR SABIO-RK; Q9AGJ6; -.
DR UniPathway; UPA00138; -.
DR GO; GO:0005737; C:cytoplasm; IEA:UniProtKB-SubCell.
DR GO; GO:0005525; F:GTP binding; IEA:UniProtKB-UniRule.
DR GO; GO:0030145; F:manganese ion binding; IEA:UniProtKB-UniRule.
DR GO; GO:0004613; F:phosphoenolpyruvate carboxykinase (GTP) activity; IEA:UniProtKB-UniRule.
DR GO; GO:0006094; P:gluconeogenesis; IEA:UniProtKB-UniRule.
DR CDD; cd00819; PEPCK_GTP; 1.
DR Gene3D; 3.40.449.10; -; 1.
DR Gene3D; 3.90.228.20; -; 1.
DR HAMAP; MF_00452; PEPCK_GTP; 1.
DR InterPro; IPR018091; PEP_carboxykin_GTP_CS.
DR InterPro; IPR013035; PEP_carboxykinase_C.
DR InterPro; IPR008209; PEP_carboxykinase_GTP.
DR InterPro; IPR035077; PEP_carboxykinase_GTP_C.
DR InterPro; IPR035078; PEP_carboxykinase_GTP_N.
DR InterPro; IPR008210; PEP_carboxykinase_N.
DR PANTHER; PTHR11561; PTHR11561; 1.
DR Pfam; PF00821; PEPCK_GTP; 1.
DR Pfam; PF17297; PEPCK_N; 1.
DR PIRSF; PIRSF001348; PEP_carboxykinase_GTP; 1.
DR SUPFAM; SSF68923; SSF68923; 1.
DR PROSITE; PS00505; PEPCK_GTP; 1.
PE 1: Evidence at protein level;
KW Cytoplasm; Decarboxylase; Direct protein sequencing; Gluconeogenesis;
KW GTP-binding; Lyase; Manganese; Metal-binding; Nucleotide-binding.
FT CHAIN 1..605
FT /note="Phosphoenolpyruvate carboxykinase [GTP]"
FT /id="PRO_0000103610"
FT ACT_SITE 273
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00452"
FT BINDING 81
FT /ligand="substrate"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00452"
FT BINDING 220..222
FT /ligand="substrate"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00452"
FT BINDING 229
FT /ligand="Mn(2+)"
FT /ligand_id="ChEBI:CHEBI:29035"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00452"
FT BINDING 249
FT /ligand="Mn(2+)"
FT /ligand_id="ChEBI:CHEBI:29035"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00452"
FT BINDING 271
FT /ligand="substrate"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00452"
FT BINDING 272..277
FT /ligand="GTP"
FT /ligand_id="ChEBI:CHEBI:37565"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00452"
FT BINDING 296
FT /ligand="Mn(2+)"
FT /ligand_id="ChEBI:CHEBI:29035"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00452"
FT BINDING 386..388
FT /ligand="substrate"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00452"
FT BINDING 388
FT /ligand="GTP"
FT /ligand_id="ChEBI:CHEBI:37565"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00452"
FT BINDING 419
FT /ligand="GTP"
FT /ligand_id="ChEBI:CHEBI:37565"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00452"
FT BINDING 514..517
FT /ligand="GTP"
FT /ligand_id="ChEBI:CHEBI:37565"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00452"
FT MUTAGEN 75
FT /note="D->A: Significantly reduced carboxykinase activity.
FT The affinity for Mn(2+), OAA and PEP decreases 9-, 2- and
FT 3-fold compared to the wild-type, respectively."
FT /evidence="ECO:0000269|PubMed:17015450"
FT MUTAGEN 75
FT /note="D->N: Significantly reduced carboxykinase activity.
FT The affinity for Mn(2+), OAA and PEP decreases 3.5-,
FT 2.8- and 3-fold compared to the wild-type, respectively."
FT /evidence="ECO:0000269|PubMed:17015450"
FT MUTAGEN 75
FT /note="D->Q: Significantly reduced carboxykinase activity.
FT The affinity for PEP and OAA decreases 9- and 2-fold
FT compared to the wild-type, respectively."
FT /evidence="ECO:0000269|PubMed:17015450"
FT MUTAGEN 75
FT /note="D->S: Significantly reduced carboxykinase activity.
FT The affinity for Mn(2+), OAA and PEP decreases 3-, 2- and
FT 3-fold compared to the wild-type, respectively."
FT /evidence="ECO:0000269|PubMed:17015450"
FT MUTAGEN 78
FT /note="D->A: Exhibits a very low activity in either the
FT OAA- or the PEP-forming direction. With Mn(2+) at 0.2 mM,
FT the specific OAA-forming activity is 0.3% that of the wild-
FT type. This value increases to 0.5%, when the Mn(2+)
FT concentration raises to 2 mM. With Mn(2+) at 0.2 mM, the
FT specific PEP-forming activity is 0.4% that of the wild-
FT type."
FT /evidence="ECO:0000269|PubMed:17015450"
FT MUTAGEN 83
FT /note="E->A: Exhibits a very low activity in either the
FT OAA- or the PEP-forming direction. With Mn(2+) at 0.2 mM,
FT the specific OAA-forming activity is 0.9% that of the wild-
FT type. This value increases to 3.4%, when the Mn(2+)
FT concentration raises to 2 mM. With Mn(2+) at 0.2 mM, the
FT specific PEP-forming activity is 2.3% that of the wild-
FT type. The affinity for PEP is not significantly different
FT from that of the wild-type, but the affinity for OAA and
FT GDP decreases 2-fold."
FT /evidence="ECO:0000269|PubMed:17015450"
SQ SEQUENCE 605 AA; 66944 MW; E07A46D4FE35EDC0 CRC64;
MTSATIPGLD TAPTKHQGLL AWVQEVAELT QPDRVVFADG SDEEYERLCA HLVEAGTFQK
LNPEKQPNSY LALSDPSDVA RVESRTFICT EREIDAGPTN NWMDPAEMRG IMTDLYRGSM
RGRTLYVVPF CMGPLDAEDP KLGVEITDSE YVVVSMRTMT RMGRAALDKL GDDGFFVKAL
HSIGAPLEPG QKDVPWPCND TKYITHFPET REIWSFGSGY GGNALLGKKC YSLRIASAMA
HDEGWLAEHM LILKLISPEN KAYFIAAAFP SACGKTNLAM LQPTIEGWRA ETVGDDIAWM
RFGKDGRLYA TNPEFGFFGV APGTNWSSNP NAMKTIAAGN TVFTNVAKTD DGDVWWEGLE
GDPQHLIDWK GNDWTPESGE KAAHPNSRYC TPISQCPTLA PEWDDPQGVP ISAILFGGRR
KTTVPLITEA RDWQHGVFIG ATLGSEQTAA AEGKVGTVRR DPMAMLPFLG YNVGDYFAHW
INVGKNADES KLPKVFFVNW FRRGDDGRFL WPGFGENSRV LKWAVERIEH KADGKSTPIG
IVPTAADLDL EGLDVDPADV DEALAVKPEE WRAELPLIEE WFEFVGEKLP TGLKDEFDAL
KERLG
