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Abstract
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.