Initial stages in the biosynthesis of porphyrins. 2. The formation of δ-aminolaevulic acid from glycine and succinyl-coenzyme A by particles from chicken erythrocytes

Abstract

Preparations of freeze-dried particles obtained from erythrocytes of anemic chickens synthesize [delta]-aminolevulic acid from succinylcoenzyme A and glycine. This is true, whether succinyl-coenzyme A is chemically synthesized or generated enzymically from succinate or [alpha]-oxoglutarate, unless diphosphopyridine nucleotide is added. It is concluded from this and other evidence that the synthesis of [delta]aminole-vulic acid from [alpha]-oxoglutarate in whole particles, described by Laver, W.G., Neuberger, A and Udenfriend, S (1958). Biochem. J. 70,4, involves the formation of succinyl-coenzyme A by the action of [alpha]-oxoglutaric dehydrogenase. The addition of pyridoxal phosphate stimulates the synthesis of [delta]-aminolevulic acid 1.5 to 3-fold. Evidence was obtained that pyridoxal phosphate is firmly bound to the enzyme. No other cofactor appears to be involved in the condensation of succinyl-coenzyme A and glycine. p-Chloromercuribenzoate, cyanide, L-penicillamine and L-cysteine inhibit the reaction strongly. D-Penicillamine, D-cysteine and glutathione inhibit slightly; iodoacetamide, sodium azide, [alpha][alpha]'' dipyridyl and carbon monoxide have no effect, while 8-hydroxyquinoline and ethy-lenediamine-tetra-acetate activate slightly. Inhibition by cyanide is unchanged in the presence of a tenfold excess of pyridoxal phosphate. Freeze-dried particles form aminoacetone on incubation with glycine, acetyl-coenzyme A and pyridoxal phosphate, and substances reacting as aminoketones when acetyl-coenzyme A is replaced by propionyl- and glutaryl-coenzyme A. Whole particles form aminoacetone from pyruvate and glycine. These reactions are slower than the synthesis of [DELTA]-aminole-vulic acid. It is suggested that the [alpha]-carbon of glycine is activated, before condensing with succinyl-coenzyme A, by the formation of a Schiff''s base between the amino group of glycine and enzyme-bound pyridoxal phosphate. The inhibition of the synthesis of [delta]-aminolevulic acid by cyanide, penicillamine and cysteine is explained on this basis.