The cytosolic isoenzyme of 3‐deoxy‐D‐arabino‐heptulosonate 7‐phosphate synthase in Spinacia oleracea and other higher plants: extreme substrate ambiguity and other properties

Abstract

The cytosolic isoenzyme of 3‐deoxy‐D‐arabino‐heptulosonate 7‐phosphate (DAHP) synthase (DS‐Co: EC 4.1.2.15) in Spinacia oleracea, Solanum tubersosum and many other higher plants was found to use a diversity of substrates. Diose (glycolaldehyde), triose (D‐glyceraldehyde, L‐glyceraldehyde and DL‐glyceraldehyde 3‐phosphate), tetrose (D‐erythrose, L‐erythrose, D‐erythrose 4‐phosphate, D‐threose and L‐threose), and pentose (D‐ribose 5‐phosphate and D‐arabinose 5‐phosphate) were utilized in combination with phosphoenolpyruvate (PEP) to make the corresponding 2‐keto‐3‐deoxy sugar acids. Glyoxylate was also utilized by DS‐Co. Glycoladehyde exhibited the highest reaction velocity when substrates were tested at 3 mM concentrations. Pentoses were poor substrates except when phsophorylated, an effect which is probably due to an increased fraction of the molecules being in the open‐chain form. Little stereoselective discrimination exists since comparable velocities were demonstrated with the D and L isomers of glyceraldehyde, erythrose or threose. The enzyme is not a reversible aldolase since pyruvate failed to substitute for PEP. The use of D‐erythrose 4‐phsophate or glycolaldehyde resulted in Km values of 1.95 mM and 8.60 mM, respectively. However, glycolaldehyde exhibited the largest VmaxKm ratio, suggesting a greater catalytic efficiency for this substrate. Glycolaldehyde is an ideal substrate for inexpensive assays of DS‐Co that are absolutely selective in the presence of two other plant enzymes which also utilize erythrose 4‐phosphate and PEP. The spinach DS‐Co enzymes required divalent metals for activity. The presence of 20 mM Mg2+, 1 mM Co2+ and 1 mM Mn2+ yielded relative activities of 100, 70 and 15, respectively. The pH optimum was 9.5 and temperature optimum for activity was 49°C. The molecular masses of DS‐Co from spinach, tobacco and pea were all in the range of 400 kDa. The possible roles of DS‐Co in biosynthesis of α‐ketoglutarate and aromatic amino acids, in biosynthesis of components of cell wall and phytotoxin, and in acting as a sink for 2‐and 3‐carbon sugars are discussed.