d-Glucose 6-Phosphate Cycloaldolase: Inhibition Studies and Aldolase Function

Abstract

D-Glucose 6-phosphate cycloaldolase is inhibited 83 % by 0.66 mM EDTA and stimulated 1.7-fold by 0.6 mM KCI. Dihy Eisenberg (4) has proposed a mechanism for the enzymatic conversion of D-glucose-6-P to 1-L-myoinositol-1-P in which an NAD+ mediated oxidation-reduction of C5 of D-glucose-6-P brackets an internal aldol condensation between Cl and C6 of the first postulated intermediate, D-xylo-5-hexulose-6-P (also referred to as 5-keto-D-glucose-6-P) as shown in thisIL-myo-Inositol 1-P His mechanism finds support in other studies (1,2,5,6,11). However, in none of the studies just referred to has it been possible to demonstrate the postulated intermediates, D-Xylo-5-hexulose-6-P and myoinosose-2 1-P. (8) showed that a lysine residue in the active site of rabbit muscle aldolase forms a Schiff base with the substrate, DHAP. Reduction of the Schiff base with NaBH4 inactivates the enzyme, leaving DHAP covalently bound to lysine. A histidine residue has also been implicated in the active center of rabbit muscle aldolase (7). Type I aldolases are maximally active over a pH range of 6.5 to 9.0, whereas type II aldolases have a much more restricted maximum, pH 7.0 to 7.5. Furthermore, type II aldolases are stimulated by K+ and are more heat labile than type I. This paper contains inhibition and inactivation studies on a plant D-glucose-6-P cycloaldolase (described also as D-glucose-6-P: 1 -L-myoinositol-l-P cyclase) which has been partially purified. The enzyme resembles a type II aldolase. MATERIALS AND METHODSIsolation and Assay of the Enzyme. D-Glucose-6-P cycloaldolase was isolated from suspension cultured cells of Acer pseudoplatanus L. as previously described (9). Active fractions obtained from gel filtration on Sephadex G-200 were used in this study. The enzyme was assayed as described (9) with slight modifications. Magnesium chloride was omitted from the reaction mixture but 4 ,umoles of MgCl2 were added during the alkaline phosphatase step (total volume, 2 ml) to increase the efficiency of the phosphatase. Since EDTA inhibits phospha-

Purification of myo-Inositol 1-Phosphate Synthase from Rice Cell Culture by Affinity Chromatography

“…Lieb et al (20) reported a cell doubling time of 22 hr at 30 C. Cells used for the present study were grown at 26 C with a cell doubling time of 33 hr.…”

“…Basic requirements for preparation of a successful affinity column include a compound which not only interacts selectively with the enzyme but which can also be covalently bound to the column matrix. Since D-glucose 6-phosphate is the substrate for MI 1-phosphate synthase, and it has been shown that the configuration about carbons 3 through 6 is required for interaction with the enzyme (26,28,30), a search was undertaken to find a derivative of D-glucose 6-phosphate which could satisfy the second condition, viz., reaction with bromoacetamidoethyl-Sepharose. P-GAT was such a compound (12).…”

Partial purification and characterization of L-myo-inositol-1-phosphate synthase of pteridophytic origin

“…D-glu cose-6-phosphate (G-6-P) ir re vers ibly isomerizes to L-myo -ino sitol-1-phos phate by L-myo-inositol-1-phosphate synthase (MIPS). The prod uct of this en zyme gen er ates free myo-inositol on dephosphorylation (Loewus and Loewus 1973). The en zyme, MIPS was iso lated and char ac ter ized from a num ber of sys tems in clud ing bac te ria (Pittner et al 1979), pro to zoa (Lohia et al 1999), lower plants (Loewus and Loewus 1971, Donahue and Henry 1981a,b, Dasgupta et al 1984, RayChoudhuri et al 1997, higher plants and an i mals (Loewus and Loewus 1983, Gumber et al 1984.…”

Myo-inositol content in pteridophytes and the isolation and characterization of L-myo-inositol-1-phosphate synthase from Diplopterygium glaucum

“…However, synthase preparations from Acer pseudoplatanus cell culture (Loewus and Loewus, 1973) and Arabidopsis thaliana (Johnson and Sussex, 1995) are inhibited by the presence of inositol.…”