CHARACTERIZATION OF THE MECHANISM OF 6-PHOSPHOGLUCONATE DEHYDROGENASE FROM TRYPANOSOMA BRUCEI AND ITS INTERACTION WITH INHIBITORS BY ISOTHERMAL TITRATION CALORIMETRY

6-Phosphogluconate dehydrogenase (6PGDH) converts 6PG to ribulose 5-phosphate and concomitantly provides NADPH, inside the pentose phosphate pathway. Its presence has been shown essential for growth of bloodstream form Trypanosoma brucei, a parasite responsible for African trypanosomiasis and it may be considered a validated drug target in this protozoan. Drugs are the principal means of intervention but new drugs are urgently needed for human African trypanosomiasis. Strong inhibitors have been found against 6PGDH, which show some selectivity versus the parasite enzyme compared the mammalian one. T. brucei 6PGDH shows only a 33% amino acid identity with the mammalian 6PGDH even if their structures have a similar overall fold and many residues nearest neighbours to the substrate are conserved. Regarding 6PGDH mechanism two residues, one acting as an acid and the other as a base, are postulated to assist all the three catalytic steps. These residues (E192 and K185 in the T. brucei) have been identified on the basis of crystallographic evidence and site-directed mutagenesis. Much remains to understand yet, for instance on which way at the end of the reaction the protonation state of the two catalytic groups changes into the opposite to that at the beginning of the reaction and on the homotropic cooperativity of the enzyme (homodimeric) and the induced enzyme changes at the substrate binding. To elucidate this and the inhibition mechanism by some lead compounds to develop as potential drugs, we have exploited isothermal titration calorimetry (ITC). ITC measurements were performed in a VP-ITC microcalorimeter and the data were fitted by nonlinear least-squares fitting using OriginTM software. The number of H+ exchanged has been measured after binding studies in different buffers. Binding was studied sometimes by fluorometry. Recombinant T. brucei 6PGDH has been purified from an overexpressing E. coli strain. Kinetics of the enzymes was studied spectrophotometrically, also in function of pH. Preparation of site-directed mutants of the enzyme has been a complementary technique to obtain information on the enzyme. cysteine reactivity has been assayed with the Ellmann reactant DTNB. Binding of the substrate and its analogues is entropy driven, while binding of coenzymes is enthalpy driven. Oxidized coenzyme and its analogue display a half-site reactivity in the ternary complex with the substrate or the inhibitors while reduced coenzyme displays full-site reactivity. Binding of 6PG and 5-phospho-ribonate (5PR) poorly affects the dissociation constant of the coenzymes, while binding of 4-phospho-erythronate (4PE), which is the most selective among the studied inhibitors for the parasite enzyme compared to the mammalian sheep liver one, decreases the dissociation constant of the coenzymes by two orders of magnitude. In a similar way the Kd of 4PE greatly decreases in the presence of the coenzymes. Results suggest that while 5PR acts as substrate analogue, 4PE mimics the transition-state of the dehydrogenation. The stronger affinity of 4PE is interpreted on the basis of the enzyme mechanism, suggesting that the inhibitor forces the catalytic K185 in the protonated state. pH curves of the mutants K185R and E192Q clearly show that other residues are involved in mechanism. It has been shown that a change from an “open” to a “closed” conformation is rate limiting. The conformational changes induced by substrate binding appear to be related with the release of about one H+/enzyme dimer, and with a drastic reduction of cysteine reactivity. The residues H188 and C372 are not directly involved in the substrate binding but are very close to the active site. The mutants H188L and C372S show a linear relationship between the residual activity and the number of H+ released and also with the cysteine reactivity. These data suggest that H188 and C372 are involved in the transition from the “open” to the “closed” form. Further studies have been performed to better characterize the mechanism of 6PGDH. Kinetic isotope effect studies on the reverse reaction, the reductive carboxylation of Ru5P to 6PG indicate that the presence of 6PG changes the rate limiting step of the reaction. In the absence of 6PG the rate limiting step is clearly identified in a conformational change of the enzyme-Ru5P complex. In the presence of 6PG this step become fast, and the rate limiting step can be identified in an isomerization of the enzyme-6PG complex according to the previously published data. These results add new support to the alternative-site model for the mechanism of 6PGDH