Purification and characterization of recombinant pyruvate phosphate dikinase from Giardia

The gene encoding pyruvate phosphate dikinase (PPDK) from Giardia duodenali s was expressed using a baculovirus system. The recombinant enzyme was puri fied 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: rev erse reaction (700 min(-1)) and the k(cat)/K-m for PPi was about two orders of magnitude higher than k(cat)/K-m for P-i, indicating that the pyruvate- forming reaction is much more efficient than the reverse, phosphoenolpyruva te (PEP)-forming process. The endogenous substrate levels found for PEP (0. 5 mM) and pyruvate (<80 mu M) support the assumption that, under physiologi cal conditions, the enzyme primarily performs a catabolic function. The mol ecular mass of the purified recombinant PPDK was analyzed by analytical ult racentrifugation and size exclusion chromatography using different assay co nditions that have been reported to affect the quaternary structure of PPDK s in other organisms. Both methods clearly indicated a dimeric structure fo r giardial PPDK with a molecular mass of about 197 kDa (monomer mass 97.6 k Da). Several compounds, primarily structural analogs of PPi, were tested fo r their ability to inhibit PPDK activity. Most of the bisphosphonates exami ned showed either no, or only a moderate, inhibitory effect on the enzyme. Imidodiphosphate was the only competitive inhibitor with respect to PPi (K- ic = 0.55 mM), whereas the bisphosphonates produced a mixed type of inhibit ion. The most active compound in inhibiting PPDK activity was oxalate, with a K-ic value of less than 1 mu M with respect to PEP. (C) 1999 Elsevier Sc ience B.V. All rights reserved.