5‐Aminolevulinic‐Acid Synthetase of Rhodopseudomonas spheroides Y

The results of initial velocity studies on the 5‐aminolevulinic acid synthetase reaction and product inhibition studies are consistent with an ordered bi‐bi mechanism in which glycine binds to the enzyme first and 5‐aminolevulinic acid dissociates last. The results are confirmed by the spectral changes of holoenzyme caused by the substrates. 5‐Aminolevulinic acid synthetase activity is inhibited only by adenosine triphosphate, guanosine triphosphate and pyrophosphate. This inhibition is competitive with glycine and non‐competitive with succinyl‐CoA. Experiments with [α‐32P]ATP, [γ‐32P]ATP and [32P2]pyrophosphate have shown that adenosine triphosphate and pyrophosphate are bound on each of both forms of 5‐aminolevulinic acid synthetase. The ratio is one mole per mole of enzyme. The estimation of –SH groups with 5,5′‐dithiobis‐(2‐nitrobenzoic acid) after binding of inhibitor shows that one –SH is masked. After p‐hydroxymercuribenzoate action on the enzyme, 5‐aminolevulinic acid synthetase does not bind anymore [32P]ATP and is completely inactive. We suggest that ATP and pyrophosphate inhibit 5‐aminolevulinic acid synthetase by acting on an –SH group of the active center of 5‐aminolevulinic acid synthetase.