An uncharacterized minor transient product, observed in our earlier studies of substrate turnover by the E48Q mutant of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase (Lee, E. H., Harpel, M. R., Chen, Y.-R., and Hartman, F. C. (1993) J. Biol. Chem. 268, 26583-26591), becomes a major product when it is trapped and stabilized with borate as an additive to the reaction mixture. Chemical characterization establishes this novel product as D-glycero-2,3-pentodiulose 1,5-bisphosphate, thereby demonstrating oxidation of the C-3 hydroxyl of D-ribulose 1,5-bisphosphate to a carbonyl. As the formation of the novel oxidation product is oxygen-dependent and generates hydrogen peroxide, its precursor must be a peroxy derivative of ribulose bisphosphate. Thus, discovery of the dicarbonyl bisphosphate lends direct support to the long standing, but heretofore unproven, postulate that the normal pathway for oxidative cleavage of ribulose bisphosphate by the wild-type enzyme entails a peroxy intermediate. Our results also suggest that stabilization of the peroxy intermediate by the wild-type enzyme promotes carbon-carbon scission as opposed to elimination of hydrogen peroxide.
Guided by comparative sequence considerations, we have examined the possibility of a catalytic role of Asp 186 of D-ribulose 5-phosphate epimerase by site-directed mutagenesis of the recombinant spinach enzyme. Accordingly, D186A, D186N, and D186E mutants of the epimerase were constructed, purified, and characterized; as judged by their electrophoretic mobilities the mutants are properly assembled into octamers like the wild-type enzyme. Based on the extent of internal quenching of Trp fluorescence, the conformational integrity of the wild-type enzyme is preserved in the mutants. The wild-type k cat of 7.1 ؋ 10 3 s ؊1 is lowered to 3.3 ؋ 10 ؊4 s ؊1 in D186A, 0.13 s ؊1 in D186N, and 1.1 s ؊1 in D186E; as gauged by D186A, altogether lacking a functional side chain at position 186, the -carboxyl of Asp 186 facilitates catalysis by >10 7 -fold. Relative to the wildtype enzyme, the K m for D-ribulose 5-phosphate is essentially unaltered with D186N and D186E but increased 10-fold with D186A. Apart from their impairments in epimerase activity, the mutants are unable to catalyze exchange between solvent protons and the C3 proton of substrates. This deficiency and the differential alterations of kinetic parameters among the mutants are consistent with Asp 186 serving as an electrophile to facilitate ␣-proton abstraction. This study is the first to identify a catalytic group of the epimerase.Despite its participation in the ubiquitous oxidative pentose phosphate pathway and its concurrent role in the reductive pentose phosphate pathway (i.e. the Calvin cycle) of photosynthetic organisms, D-ribulose 5-phosphate 3-epimerase (EC 5.1.3.1), which catalyzes the interconversion of Ru5P 1 and Xu5P, has not been well-characterized with respect to structure-function relationships. This relative neglect is probably a consequence of low natural abundance of the epimerase and, in the case of the plant enzyme, pronounced instability. We have recently removed these barriers to detailed structural and mechanistic studies by designing a heterologous overexpression system for the gene that encodes spinach chloroplast Ru5P epimerase and by devising a facile purification scheme for the recombinant enzyme that is compatible with its lability (1).The epimerase-catalyzed reaction probably entails a twobase mechanism with an enediolate intermediate (Fig. 1). This supposition is based on the observations that Ru5P formed from [3-2 H]Xu5P was completely free of deuterium, even though deuterium was completely retained in the remaining Xu5P (2). Later findings (3) that isomerization of D-erythrose 4-phosphate to D-erythrulose 4-phosphate by Ru5P epimerase proceeded by intramolecular proton transfer, whereas the C2 proton of D-threose-4-phosphate formed concurrently by epimerization of D-erythrose-4-phosphate was derived entirely from water, lends additional credence to a two-base mechanism. In Fig. 1, we invoke a general acid to facilitate ␣-proton abstraction and to stabilize the enediolate intermediate based merely on the prevalence of such group...
A nucleoside triphosphatase (NTPase) present in highly purified preparations of pea nuclei was partially characterized. The activity of this enzyme was stimulated by divalent cations (Mg2" = Mn2" > Ca2"), but was not affected by the monovalent cations, Na' and K+. The Mg2"-dependent activity was further stimulated by concentrations of Ca2" in the low micromolar range. It could catalyze the hydrolysis of ATP, GTP, UTP, and CTP, all with a pH optimum of 7.5.
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