Cysteine 365 may have a role in the catalytic mechanism of 6-Phosphogluconate Dehydrogenase from Sheep Liver

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 (Ru5P) with the concomitant reduction of NADP to NADPH (1,2). A general base/general acid mechanism has been suggested on the basis of pH dependence of kinetic parameters. Recent site-specific mutagenesis studies have indicated the Lysine 183 (K183) and the Glutammate 190 (E190) as best candidate for the role of catalytic base and acid, respectively (2,3). Briefly: K183 accepts a proton from the 3-hydroxyl of 6PG concomitant with hydride transfer leading the reaction to the formation of 3-keto-6PG intermediate. The latter molecule is then decarboxylated to the enediol of Ru5P, with K183 donating back the proton to C-3 of 3-Keto. Finally, E190 and K183 assist the tautomerization of the enediol to Ru5P, with the first residue that donates a proton to C1 and the second one that accepts a proton from C2 of the enediol. The crystal structure of the apoenzyme shows K183 protonated, whilst in the complex with the substrate it appear unprotonated (5). The missing proton is supposed to be accepted from a not well recognized residue which presumably may play a an relevant role in the catalytic mechanism of the enzyme. We believe that this process may be assisted by a Cysteine found abnormally reactive only in absence of substrate. We suggest that the most probable Cysteine, amongst the seven of 6PGDH, may be the C372 because it is the nearest to K183 and it is conserved in all sequences of the enzyme. Due to the proximity of Hystidine 186 to the two residues, and the high ionization reversibility of its side-chain, it may be hypothesized that the Hystidine could act as bridge in the exchange of the proton between K183 and C372. We change C365 in a Serine,a Valine and a Aspartate in order to evaluate if the function of the residue is crucial in the catalysis of 6PGDH. MATERIAL AND METHODS: Three mutations for the Cys 365 (Serine, Valine and Aspartate) were obtained by a in vitro site-specific mutagenesis system performed on the 6PGDH recombinant gene. All three resulting 6PGDH cDNA containing the mutation were cloned into a pQE-30 plasmid in the host strain M15[pREP4]. Each mutation was verified by sequence analysis. All mutant proteins 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 pH dependence of kinetic parameters was assessed using three different buffer, in order to cover a pH range from 4.0 to 9.0. The primary deuterium isotope effects were determined fluorimetrically measuring the reduced coenzyme fluorescence at 460 nm, with excitation at 340 nm. RESULTS: The substitutions of C365 with Aspartate, Serine and Valine cause a decline in V/Et by at least 2 order of magnitude with respect to WT. On the contrary, the affinity constants, for both coenzyme (NADP) and substrate, do not show marked differences if compared to that of WT. Nevertheless, a small tendency in improving the affinity of 6PG has been observed (mostly in C365S where a KM three times lower has been found). The kinetic parameters (V; V/KNADP ; V/K6PG) of each mutant is pH-dependent as it occurs in WT, decreasing at low and high pH with slopes of 1 and –1, respectively. Thus, two independent pK, one in the acid side of the pH profile the other in the basic side, can be obtained by fitting the experimental data. The calculated values remarkably differ from those of WT (the pKb of the Serine mutant is around 1.0 pH) indicating that the substitution of 365C with the three amminoacids have perturbated the ionization constant of both catalytic residues of the enzyme. These data are consistent with a crucial role of C365 in modulating the pK of K183, which during the catalysis alternately acts as base and acid. The function of the residue may be that of shuttling the proton with the probable mediation of the proximal H183. The ability of this Hystidine to allow the transfer of the proton from inside to outside, and inversely, of catalytic system is going to be studied by further mutagenesis experiments.