delta-Aminolevulinate dehydratase inhibition by 2,3-dimercaptopropanol is mediated by chelation of zinc from a site involved in maintaining cysteinyl residues in a reduced state.

The mechanisms underlying mouse delta-aminolevulinate dehydratase (ALA-D) inhibition by a chelating agent used in the treatment of heavy metal poisoning, 2,3-dimercaptopropanol (British Anti-Lewisite), were investigated. ALA-D inhibition by 2,3-dimercaptopropanol was totally reversed by 25-100 microM Zn2+, indicating that inhibition was due to chelation of zinc by 2,3-dimercaptopropanol. Our data suggested that zinc bound to a labile site (displaced by 25-40 microM EDTA or 500 microM 2,3-dimercaptopropanol) is involved in maintaining the sulfhydryl groups of ALA-D in a reduced state (essential for enzyme activity), since inhibition by these compounds was reversed by 10 mM dithiotreitol (a reducing agent). On the other hand, 10 mM dithiotreitol did not reverse ALA-D inhibition by a higher concentration of EDTA (100 microM). Accordingly, 2,3-dimercaptopropanol appears to inhibit ALA-D through a mechanism similar to that of low EDTA concentrations. Neither oxidized 2,3-dimercaptopropanol nor reactive oxygen species appeared to contribute for ALA-D inhibition by reduced 2,3-dimercaptopropanol. Taken together, these results suggest that 2,3-dimercaptopropanol inhibits ALA-D by chelating Zn2+ from a labile site that is involved in maintaining enzyme sulfhydryl groups in a reduced state. This site is compatible with the ZnB or Zn beta previously described in mammalian and bacterial ALA-D.

delta-Aminolevulinate dehydratase inhibition by ascorbic acid is mediated by an oxidation system existing in the hepatic supernatant.

The effect of ascorbic acid (AA) on hepatic delta-aminolevulinic acid dehydratase (ALA-D) activity was studied. AA decreased enzyme activity by reducing maximum velocity and tended to increase the Michaelis constant. ALA-D inactivation by AA occurred similarly both in air and argonium atmosphere incubation. DTT reduced considerably the inhibitory effect of AA on ALA-D, but glutathione was ineffective in reversing inactivation. These data indicate that inhibition occurs mainly due to an acceleration of the oxidation rate mediated by the hepatic supernatant utilizing AA in sulfhydryl groups of cysteine residues present at the ALA-D active site. AA probably acts on cysteine from the ALA-D B site since cucumber and radish leaves ALA-D was not inhibited by AA (up to 16 mM). The addition of free radical scavengers to the medium did not alter ALA-D inactivation caused by AA, indicating that active oxygen species formed during AA oxidation were not directly related to -SH oxidation. The chelation of zinc ions from the enzyme by EDTA turned ALA-D more susceptible to the inhibitory effect of AA. This effect seems to involve mainly ZnB, which is known to bind to four cysteines. The present data suggest that AA may participate in the regulation of the heme biosynthesis pathway by promoting a reversible inactivation of ALA-D.