INTRODUCTION: 6-Phosphogluconate Dehydrogenase (6PGDH) is the third enzyme in the Pentose Phosphate Pathway and catalyzes the oxidative decarboxylation of 6-phosphogluconate (6PG) to ribulose 5-phosphate with the concomitant reduction of NADP to NADPH. Kinetic studies suggested a significant role played by the molecule of NADPH in the decarboxylation step of the catalytic process of 6PGDH(1). Furthermore, a comparison between the crystal structure of the binary complex of E:NADP and that of E:NADPH (2) showed a evident shift of nicotinamide ring upon reduction towards the residues directly involved in the catalysis. This different location of the active group of NADPH could affect the ionic environment of the active site and consequently influence the process of catalysis. The above mentioned crystallographic data displayed the Methionine 13 being in a crucial position for determining the optimal orientation of nicotinamide ring of NADPH. MATERIAL AND METHODS: Five mutations for the Met 13 (Ile, Cys, Gln, Val and Phe) were obtained by a in vitro site-specific mutagenesis system performed on the 6PGDH recombinant gene. All five resulting 6PGDH cDNA containing the mutation were cloned into a pQE-30 plasmid in the host strain M15[pREP4].All the mutations were verified by sequence analysis. All mutants were purified with a Ni-NTA resin, taking advantage of a six-His tag added to the N-terminus all 6PGDH derivatives. Neither CD nor fluorimetric analysis revealed evident difference in the gross structure of the mutant enzymes with respect to that of WT. Initial velocity pattern were performed spectrophotometically measuring the appearance of NADPH at 340 nm in the direction of oxidative decarboxylation. The primary deuterium isotope effects were determined fluorimetrically measuring the reduced coenzyme fluorescence at 460 nm, with excitation at 340 nm. RESULTS: The kinetic parameters obtained for the mutants showed that substitution of Methionine 13 with other residues caused comparable decline in affinity for the coenzyme with respect to that of WT. Indeed for all mutants a 10-fold increase of KM for NADP was observed (except for the KM of Cys mutant which is just 3-fold higher). As expected, since the residue we substituted was located in the NADP binding site, no change in affinity for the substrate 6PG were revealed. Only for the Cys and Phe mutants we detected a 10-fold decrease in the turnover number (V/ET). The rest of mutants showed the same value of V/ET measured for WT. In order to investigate if any variation in kinetic mechanism of the 6PGDH occurred upon substitution of residue in position 13, primary deuterium isotope effect were measured. The magnitude of VD and (V/K)D for both substrates for Phe and Cys mutants were equal to 1 (within experimental error), while Iso and Val mutants evidenced identical values to that of WT (VD=(V/K)D=1.8) (3). On the contrary Gln was found to have an isotope effect for the first and the second order constant around 2.5. The absence of isotope effect of Cys and Phe mutant could be interpreted with an inversion of the rate-limiting step from the dehydrogenation to another one, most probably the decarboxylation step. Alike, the increase of VD and (V/K)D of the Gln mutant could be explained by a further decrease of the rate of dehydrogenation step which could become more ratelimiting in overall catalysed reaction. To obtain further and definitive proofs in support of this mechanism, multiple isotope effect measurements will be performed.

Role of Methionine 13 in the catalytic mechanism of 6-phosphogluconate dehydrogenase from sheep liver

CERVELLATI, Carlo;GAMBETTI, Stefania;DALLOCCHIO, Franco Pasquale Filippo
2003

Abstract

INTRODUCTION: 6-Phosphogluconate Dehydrogenase (6PGDH) is the third enzyme in the Pentose Phosphate Pathway and catalyzes the oxidative decarboxylation of 6-phosphogluconate (6PG) to ribulose 5-phosphate with the concomitant reduction of NADP to NADPH. Kinetic studies suggested a significant role played by the molecule of NADPH in the decarboxylation step of the catalytic process of 6PGDH(1). Furthermore, a comparison between the crystal structure of the binary complex of E:NADP and that of E:NADPH (2) showed a evident shift of nicotinamide ring upon reduction towards the residues directly involved in the catalysis. This different location of the active group of NADPH could affect the ionic environment of the active site and consequently influence the process of catalysis. The above mentioned crystallographic data displayed the Methionine 13 being in a crucial position for determining the optimal orientation of nicotinamide ring of NADPH. MATERIAL AND METHODS: Five mutations for the Met 13 (Ile, Cys, Gln, Val and Phe) were obtained by a in vitro site-specific mutagenesis system performed on the 6PGDH recombinant gene. All five resulting 6PGDH cDNA containing the mutation were cloned into a pQE-30 plasmid in the host strain M15[pREP4].All the mutations were verified by sequence analysis. All mutants were purified with a Ni-NTA resin, taking advantage of a six-His tag added to the N-terminus all 6PGDH derivatives. Neither CD nor fluorimetric analysis revealed evident difference in the gross structure of the mutant enzymes with respect to that of WT. Initial velocity pattern were performed spectrophotometically measuring the appearance of NADPH at 340 nm in the direction of oxidative decarboxylation. The primary deuterium isotope effects were determined fluorimetrically measuring the reduced coenzyme fluorescence at 460 nm, with excitation at 340 nm. RESULTS: The kinetic parameters obtained for the mutants showed that substitution of Methionine 13 with other residues caused comparable decline in affinity for the coenzyme with respect to that of WT. Indeed for all mutants a 10-fold increase of KM for NADP was observed (except for the KM of Cys mutant which is just 3-fold higher). As expected, since the residue we substituted was located in the NADP binding site, no change in affinity for the substrate 6PG were revealed. Only for the Cys and Phe mutants we detected a 10-fold decrease in the turnover number (V/ET). The rest of mutants showed the same value of V/ET measured for WT. In order to investigate if any variation in kinetic mechanism of the 6PGDH occurred upon substitution of residue in position 13, primary deuterium isotope effect were measured. The magnitude of VD and (V/K)D for both substrates for Phe and Cys mutants were equal to 1 (within experimental error), while Iso and Val mutants evidenced identical values to that of WT (VD=(V/K)D=1.8) (3). On the contrary Gln was found to have an isotope effect for the first and the second order constant around 2.5. The absence of isotope effect of Cys and Phe mutant could be interpreted with an inversion of the rate-limiting step from the dehydrogenation to another one, most probably the decarboxylation step. Alike, the increase of VD and (V/K)D of the Gln mutant could be explained by a further decrease of the rate of dehydrogenation step which could become more ratelimiting in overall catalysed reaction. To obtain further and definitive proofs in support of this mechanism, multiple isotope effect measurements will be performed.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/531324
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