Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase.
This work reports the first functional characterization of leishmanial PEPCK. The recombinant Leishmania major enzyme (Lmj_PEPCK) exhibits equivalent kcat values for the phosphoenolpyruvate (PEP) and oxaloacetate (OAA) forming reactions. The apparent Km towards OAA is 10-fold lower than that for PEP, while the Km values for ADP and ATP are equivalent. Mutagenesis studies showed that D241, D242 and H205 of Lmj_PEPCK like the homologous residues of all known PEPCKs are implicated in metal ions binding. In contrast, the replacement of R43 for Q nearly abolishes Lmj_PEPCK activity. Moreover, the Y180F variant exhibits unchanged Km values for PEP, Mn(2+), and [Formula: see text] , being the kcat for PEP- but not that for OAA-forming reaction more notably decreased. Instead, the Y180A mutant displays an increase in the Km value towards Mn(2+). Therefore in Lmj_PEPCK, Y180 seems to exert different functions to those of the analogous residue in ATP- and GTP-dependant enzymes. Besides, the guanidinium group of R43 appears to play an essential but yet unknown role. These findings promote the need for further structural studies to disclose whether Y180 and R43 participate in the catalytic mechanism or/and in the transitions between the open and the catalytically competent (closed) forms of Lmj_PEPCK.
Instituto de Química y Fisicoquímica Biológica IQUIFIB-CONICET, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, C1113AAD, Buenos Aires, Argentina.
Máximo Hernán
MH
Sosa
Instituto de Química y Fisicoquímica Biológica IQUIFIB-CONICET, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, C1113AAD, Buenos Aires, Argentina.
Lucila
L
Giordana
Instituto de Química y Fisicoquímica Biológica IQUIFIB-CONICET, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, C1113AAD, Buenos Aires, Argentina. Electronic address: cnowicki@qb.ffyb.uba.ar.
Cristina
C
Nowicki
D000595:Amino Acid Sequence; D055162:Biocatalysis; D018320:Leishmania major / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D019764:Phosphoenolpyruvate Carboxykinase (ATP)
3-Mercaptopicolinic acid; Leishmania parasites; Mutagenesis studies; Phosphoenolyruvate carboxykinase