-Aminolevulinic Acid-Synthesizing Enzymes Need an RNA Moiety for Activity
-Aminolevulinic acid (ALA) is a ratelimiting factor of the chlorophyll biosynthetic pathway. It is synthesized from glutamate in plastids and is the first committed intermediate of the chlorophyll biosynthetic pathway (1). The enzymes from barley plastids (2) and Chlamydomonas cells (3) that are responsible for the formation of ALA from glutamate have been isolated and fractionated into three parts by serial affinity chromatography with a Blue Sepharose column and a heme-Sepharose column. The fractions are designated as Blue Sepharose-bound (B), heme-Sepharose-bound (H), and runoff (R). When combined together, the fractions can convert glutamate to ALA, but they are inactive when assayed alone. Cofactors required are adenosine triphosphate (ATP), Mg2 , and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) (2, 3).
The best supported scheme for ALA formation from glutamate is shown in Fig. 1. It had been proposed that glutamate is activated by a kinase using ATP and Mg2 to form glutamate-1-phosphate. Subsequently, a dehydrogenase converts glutamate-1-phosphate to glutamate-1-semialdehyde (GSA). Finally, GSA is converted to ALA (4). GSA has been synthesized from N-carbobenzoxy-glutamatic acid- -benzyl ester (4), and the structure of its acetal has been verified (5). The enzyme GSA aminotransferase (E.C. 22.214.171.124), which converts GSA to ALA, has been purified from barley plastids (6). Since none of the other intermediates have been isolated and identified, the exact nature of the proposed reaction sequence remains unclear. However, we have shown that (i) GSA aminotransferase is in the R fraction (2, 3); (ii) the H and B fractions together convert glutamate to a compound that purifies with synthesized GSA on high-performance liquid chromatography (2); and (iii) the compound synthesized by the H and B fractions from glutamate, as well as the synthesized GSA, can be converted by the R fraction to ALA (2, 3). This ALA was identified by high-performance liquid chromatography and thin-layer chromatography against authentic samples of ALA.
Under the assumption that H and B fractions together can convert glutamate to GSA, we have been studying the components of the ALA-synthesizing enzymes in either H or B fractions by the following assay. We combine the H fraction with the B fraction and add radioactive glutamate plus all the cofactors. After incubation, radioactive GSA is purified, and the radioactive label is counted (3). Since we have been able to recover GSA quantitatively with our purification procedure (2, 3), this is considered a reliable and...