The enzyme which catalyzes the conversion of D-erythrose 4-phosphate to D-erythrulose 4-phosphate and D-threose 4-phosphate has been purified to homogeneity from a crude extract of beef liver. Analysis of the purified enzyme by Sephadex G-100 gel filtration and sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed it to be a dimer of relative molecular mass 43000. From the gas chromatography/mass spectrometry analyses of the enzymatic reaction products, it appeared that about 90% of the total amount of tetrose 4-phosphate was present as D-erythrulose 4-phosphate after equilibration. The purified enzyme, which is tentatively called 'erythrose-4-phosphate isomerase' had no significant isomerase activities on D-glyceraldehyde 3-phosphate, D-ribose 5-phosphate, D-glucose 6-phosphate and D-fructose 6-phosphate, but a strong ~-ribulose-5-phosphate 3-epimerase activity was co-purified with the erythrose-4-phosphate isomerase activity through every step in the isolation. Both the erythrose-4-phosphate isomerase and ~-ribulose-5-phosphate 3-epimerase activities were inactivated at the same rate at the elevated temperature, and also inhibited to the same extent by various inhibitors. It is likey, that both activities are catalyzed by the single enzyme protein.We have reported previously the purification and properties of D-erythrulose reductase (EC 1.1.1.1 62) [I, 21 and aldehyde reductase (EC 1.1.1.2) [3], which catalyze the reduction of D-erythrulose and D-erythrose respectively. These enzymes were considered to be important with respect to the tetrose metabolism. In the continuing efforts from this laboratory to understand how the naturally occurring tetritols, such as erythritol and threitol excreted in urine, are synthesized we recently found an enzyme catalyzing both isomerization and epimerization of D-erythrose 4-phosphate (Ery4P) to D-erythrulose 4-phosphate and D-threose 4-phosphate in beef liver [4]. This enzyme could be described as erythrose-4-phosphate (Ery4P) isomerase since it was able to catalyze the isomerization of Ery4P to the major product, D-erythrulose 4-phosphate. Since other mammalian enzymes catalyzing such a reaction have not been reported until now, this enzyme has been the subject of extensive research by our laboratory.In the present report we describe the characterization of a highly purified enzyme having both the Ery4P isomerase and epimerase activities from beef liver, and discuss the mechanism

An enzyme preparation from beef liver catalyzed the isomerization and epimerization of D-erythrose 4-phosphate to D-erythrulose 4-phosphate and D-threose 4-phosphate. The presence of D-erythrulose 4-phosphate and D-threose 4-phosphate was demonstrated by several analytical methods. After dephosphorylation, the presence of Derythrulose and D-threose was confirmed by thin-layer chromatography, gas-liquid chromatography and an enzymatic method depending upon werythrulose reductase. The enzymatic products were also identified and simultaneously quantitated by a new procedure using gas chromatography/mass spectrometry. Each of three tetroses was distinguished by the combination of the reduction with sodium borodeuteride and the determination of relative intensities of the ion pairs m / z 379 and 380 of sugar tetritol trifluoroacetate. By gas chromatography/mass spectrometry, we observed that D-threose 4-phosphate was also converted into D-erythrulose 4-phosphate and Derythrose 4-phosphate. At the equilibrium, about 90 "/, of the tetrose 4-phosphate existed in the form ofD-erythrulose 4-phosphate. On the basis of gas chromatography/mass spectrometric evidence together with gas chromatographic and thin-layer chromatographic patterns, it is suggested that the single enzyme of the beef liver catalyzed both reactions of isomerization and epimerization of aldotetrose 4-phosphate.It has been known that tetritols such as erythritol and threitol are excreted in relatively large amounts in urine [l -31, but the metabolic pathways for the formation of the tetritols were unclear. In the course of our studies on tetrose metabolism, Uehara et al. have found two enzymes which were assumed to play a role in the reductive metabolism of tetroses. One of them is aldehyde reductase (EC 1 .l. In this paper we report that Ery4P is converted into Derythrulose 4-phosphate and D-threose 4-phosphate by a beef liver enzyme. We also describe a new practical method combined with gas chromatography/mass spectrometry (GC/MS), by which Ery4P, D-erythrulose 4-phosphate, and uthreose 4-phosphate in the reaction mixture can be quantitatively determined.Ahhwviutions. Ery4P, D-erythrose 4-phosphate; NaBH,, sodium borohydride; NaBD,, sodium borodeuteride; Bistris, 2-[bis(2-hydroxyethyl) amino]-2-(hydroxymethyl)-propane-l,3-diol; ECTEOLA, epichlorohydrin triethanolamine; GLC, gas-liquid chromatography; TLC, thin-layer chromatography; GC/MS, gas chromatography/mass spectrometry.Enzymes. Aldehyde reductase (EC 1.1.1.2); D-erythrulose reductase (EC1.1.1.162); acid phosphatase (EC3.1.3.2). MATERIALS A N D METHODS MaterialsD-Erythrose 4-phosphate (Ery4P) and wthreose 4-phosphate were prepared from D-glucose 6-phosphate and Dgalactose 6-phosphate, respectively, by a slight modification of the method of Simpson et al. [9]. D-Erythrose 4-phosphate was also purchased from Sigma Chemical Co. D-Erythrose and Dthreose were prepared from D-glucose and wgalactose, respectively, by the method of Perlin and Brice [lo], and purified extensively by a combined procedure of ...