Purification and characterization of recombinant pyruvate phosphate dikinase from Giardia.
The gene encoding pyruvate phosphate dikinase (PPDK) from Giardia duodenalis was expressed using a baculovirus system. The recombinant enzyme was purified to homogeneity and its enzymological and solution structure properties characterized. The catalytic constant for the pyruvate-producing reaction was about twice as high (1560 min(-1) at 30 degrees C) as that for the reverse reaction (700 min(-1)) and the k(cat)/Km for PPi was about two orders of magnitude higher than k(cat)/Km for Pi, indicating that the pyruvate-forming reaction is much more efficient than the reverse, phosphoenolpyruvate (PEP)-forming process. The endogenous substrate levels found for PEP (0.5 mM) and pyruvate (< 80 microM) support the assumption that, under physiological conditions, the enzyme primarily performs a catabolic function. The molecular mass of the purified recombinant PPDK was analyzed by analytical ultracentrifugation and size exclusion chromatography using different assay conditions that have been reported to affect the quaternary structure of PPDKs in other organisms. Both methods clearly indicated a dimeric structure for giardial PPDK with a molecular mass of about 197 kDa (monomer mass 97.6 kDa). Several compounds, primarily structural analogs of PPi, were tested for their ability to inhibit PPDK activity. Most of the bisphosphonates examined showed either no, or only a moderate, inhibitory effect on the enzyme. Imidodiphosphate was the only competitive inhibitor with respect to PPi (Kic = 0.55 mM), whereas the bisphosphonates produced a mixed type of inhibition. The most active compound in inhibiting PPDK activity was oxalate, with a Kic value of less than 1 microM with respect to PEP.
Hiltpold
A
A
Institute of Parasitology, University of Zürich, Switzerland.
Thomas
R M
RM
Köhler
P
P
D000818:Animals; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics; D002460:Cell Line; D004164:Diphosphonates / Q000378:metabolism; D005872:Giardia / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D011772:Pyruvate, Orthophosphate Dikinase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity
D004164:Diphosphonates; D011994:Recombinant Proteins; D011772:Pyruvate, Orthophosphate Dikinase