Delta-aminolevulinic acid dehydratase (ALA-D, EC 4.2.1.24) is a sulfhydryl-containing enzyme that asymetrically condenses two molecules of delta-aminolevulinic acid (ALA), catalyzing the formation of porphobilinogen, the monopyrrole precursor of ali biological tetrapyrroles (corrins, porphyrins, chlorins). The two ALA molecules have been termed Aside ALA and P-side ALA in reference to their fates as the acetyl and propionyl halves of the product. P-side ALA binds first and forms a Schiffbase with an active-site Iysine. ALA-D is a cytosolic enzyme present in mammals, plants, fungi and bacteria. Bovine enzyme has a molecular mass of 280 000 Da and is composed of eight similar subunits of 35 000 Da, but only four of the subunits form a Schiff-base with the substrate (half-site reactivity). ALA-D from all organisms requires a bivalent metal ion for activity. Although the considerable sequence conservation among ALA-D enzyme from various organisms, there are species-dependent differences in metal ion requirements for enzyme activity. ALA-D is a zinc-dependent enzyme in animals, yeast and some bacteria. Mammalian enzyme bounds 8 zinc ions/octamer. Bovine ALA-D contains two types of Zn2+ binding sites (A and B), each at a stoichiometry of four per octamer. A-metal-ion-binding sites, with a single cysteine residue among its ligands, bind the four zinc ions essential for ALA-D activity (catalytic zinc), which plays a role in A-side ALA binding, in inter-ALA bond formation and in product binding. B-metal-ion-binding sites, with four cysteine residues among its ligands, bind zinc ions refered to as structural, which seems to be involved in the protection of sulfhydryl groups from oxidation. An A-zinc-ion-binding site has been proposed to be present at a number of four per octamer on the enzyme from plants, but has not been demonstrated yet. ALA-D from plants contains two types of magnesium binding sites: four B-metal-ion-binding sites (bind magnesium essential for ALA-D activity) and eight C-metal-ion-binding sites (bind magnesium that activates the enzyme but is not essential for activity). The cysteine-rich sequence of mammalian ALA-D that presumably corresponds to the B-metalion-binding site is replaced by an aspartate-rich sequence in plant ALA-D, probably accounting for the difference on metal-ion requirement (Mg2+ instead of Zn2+ on B-metal-ion-binding site from plant ALA-D). E. coli ALA-D binds eight Zn2+ (presumably four at A-metal-ion-binding site and four at B-metalion-binding site) and eight Mg2+ (presumably at C-metal-ion-binding site) per octamer. Due to its sulfhydrilic nature ALA-D is inhibited by heavy metals such as lead and mercury, serving as a measure of metal intoxication. In addition the inhibition of this enzyme has been implicated with pathological changes observed in some types of porphyrias, hepatorenal tyrosinemia and after lead or mercury exposure. ALA-D inhibition may impair haeme biosynthesis and leads to ALA accumulation, which besides being a potent agonist of y-aminobutiric acid autoreceptors may act as a prooxidant.
Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa
