METAL REQUIREMENTS OF THE ENZYMES CATALYZING CONVERSION OF GLUTAMATE TO DELTA-AMINOLEVULINIC-ACID IN EXTRACTS OF CHLORELLA-VULGARIS AND SYNECHOCYSTIS SP PCC-6803

In the biosynthetic conversion of glutamate to the tetrapyrrole precur sor, delta-aminolevulinic acid (ALA), glutamate is activated at C-1 by glutamyl-tRNA synthetase-catalyzed ligation to tRNA(Glu). Glutamyl-tR NA reductase next catalyzes reduction of the activated glutamate to gl utamate-1-semialdehyde (GSA), which is then converted to ALA by GSA am inotransferase. Glutamyl-tRNA synthetase is known to require a divalen t metal (usually Mg2+) for activity, but it has not been established w hether Mg2+ or another metal ion is also required for glutamyl-tRNA re ductase or GSA aminotransferase, because these enzymes have previously been assayed in combined incubations containing all factors required for conversion of glutamate to ALA. We now report the metal requiremen ts individually for each of the three enzyme reactions. Glutamyl-tRNA reductase activity in extracts from both Chlorella vulgaris and Synech ocystis sp. PCC 6803 was stimulated by Mg2+ and inhibited by EDTA. EDT A-pretreated Chlorella glutamyl-tRNA reductase-containing fraction had very little activity in the absence of added Mg2+, but recovered full activity in incubations containing added Mg2+. The divalent metal req uirement could be met by Mg2+, Mn2+, or Ca2+. Maximum activity was rea ched at approximately 15 mM concentration of each of these metals, and higher concentrations were inhibitory. Zn2+ was inhibitory at micromo lar concentrations. Chlorella glutamyl-tRNA synthetase showed a metal requirement that could be met by Mg2+ or Mn2+ but not Ca2+. Maximum ac tivity was reached at approximately 15 mM Mg2+ or Mn2+. Although the p resence of 10 mM Ca2+ did not affect the Mg2+ concentration optimum, C a2+ increased the effectiveness of low concentrations of Mg2+. In cont rast to glutamyl-tRNA synthetase and glutamyl-tRNA reductase, Chlorell a GSA aminotransferase did not show a metal requirement or inhibition by EDTA. However, EDTA decreased nonenzymatic transformation of GSA to ALA. (C) 1994 Academic Press, Inc.