Characterization of amyloplastic phosphohexose isomerase from immature wheat (Triticum aestivum L.) endosperm.

Phosphohexose isomerase from amyloplasts of immature wheat endosperm was purified 133-fold. The enzyme had a molecular weight of 130 kDa and maximum activity at pH 8.6. It showed normal hyperbolic kinetics for both fructose-6-P and glucose-6-P with Km of 0.12 mM and 0.44 mM, respectively. pH had a great influence on Km for fructose-6-P. Using glucose-6-P as the substrate, the equilibrium was reached at 23% fructose-6-P and 77% glucose-6-P and an equilibrium constant of about 3.0. The delta F calculated from the apparent equilibrium constant was +742 cal.mol-1. The activation energy calculated from the Arrhenius plot was 7450 cal.mol-1. None of the sulphydryl reagents at 2.5 mM concentration inactivated the enzyme. The enzyme was competitively inhibited by 6-phosphogluconate, ribose-5-P and ribulose-5-P with Ki values of 0.18, 0.14, and 0.13 mM, respectively. The probable role of the enzyme in starch biosynthesis in amyloplasts is discussed.

Characterization of cytosolic phosphoglucoisomerase from immature wheat (Triticum aestivum L.) endosperm.

Phosphoglucoisomerase from cytosol of immature wheat endosperm was purified 650-fold by ammonium sulphate fractionation, isopropyl alcohol precipitation, DEAE-cellulose chromatography and gel filtration through Sepharose CL-6B. The enzyme, with a molecular weight of about 130,000, exhibited maximum activity at pH 8·1. It showed typical hyperbolic kinetics with both fructose 6-P and glucose 6-P with Km of 0·18 mM and 0·44mM respectively. On either side of the optimum pH, the enzyme had lower affinity for the substrates. Using glucose 6-P as the substrate, the equilibrium was reached at 27% fructose 6-P and 73% glucose 6-P with an equilibrium constant of 2·7. The ΔF' calculated from the apparent equilibrium constant was +597 cal mol–1 . The activation energy calculated from the Arrhenius plot was 5500 cal mol–1. The enzyme was completely inhibited by ribose 5-P, ribulose 5-P and 6-phosphogluconate, with Ki values of 0·17, 0·25 and 0·14 mM respectively. The probable role of the enzyme in starch biosynthesis is discussed.