Heptulose Synthesis from Nonphosphorylated Aldoses and Ketoses by Spinach Transketolase

Abstract

Abstract 1. The enzyme present in spinach leaves that catalyzes the transfer of a 2-carbon fragment from hydroxypyruvate to d-ribose, l-lyxose, d-xylose, and l-arabinose to form the corresponding free heptuloses was purified to near electrophoretic homogeneity and found to be identical with transketolase. The maximum velocity for the reaction of these pentoses with a saturating concentration of hydroxypyruvate was the same, whereas the Michaelis constants were different. The Michaelis constants were 45 mm for d-ribose, 55 mm for l-lyxose, 120 mm for l-arabinose, and 230 mm for d-xylose. It may thus be possible to account for the production of free heptuloses in plants which have been infiltrated with pentoses, without invoking the participation of phosphorylated pentose intermediates or of a new ligase. 2. Also serving as donors with transketolase when ribose was the acceptor were d-fructose and l-sorbose. When unlabeled d-sedoheptulose and ribose-5-3H were incubated with the enzyme, sedoheptulose-7-3H was formed, showing that sedoheptulose was also a donor. Moreover, 14C-gluco-heptulose was formed from sedoheptulose and l-arabinose-1-14C. 3. The molecular weight of transketolase was estimated as 100,000 ± 10,000 from gel filtration, electrophoresis in sodium dodecyl sulfate on polyacrylamide, and sedimentation equilibrium experiments.