Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase.

SOSA MÁXIMO,; GIORDANA LUCILA,; NOWICKI , CRISTINA

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

ELSEVIER SCIENCE INC

Lugar: Amsterdam; Año: 2015 vol. 583 p. 120 - 129

0003-9861

This work reports thefirst 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 andH205 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 unchangedKmvalues for PEP, Mn2+, and HCO3 , being the kcatfor PEP- but notthat for OAA-forming reaction more notably decreased. Instead, the Y180A mutant displays an increase in the Kmvalue towards Mn2+. 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. Thesefindings 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_PEPC