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 |
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Authors: | Ron Lou Doong John E. Gander Robert J. Ganson Roy A. Jensen |
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Affiliation: | Dept of Microbiology and Cell Science, Univ. of Florida, Gainesville, FL 32611, USA. |
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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. |
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Keywords: | Aromatic biosynthesis DAHP synthase glycoladehyde pea Pisum sativum potato Solanum tuberosum spinach Spinacia oleracea substrate ambiguity |
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