PMID:8439296
| Citation |
Spencer, P and Jordan, PM (1993) Purification and characterization of 5-aminolaevulinic acid dehydratase from Escherichia coli and a study of the reactive thiols at the metal-binding domain. Biochem. J. 290 ( Pt 1):279-87 |
|---|---|
| Abstract |
5-Aminolaevulinic acid dehydratase (ALAD) from a recombinant strain of Escherichia coli was purified to homogeneity. The enzyme is a homo-octamer of subunit M(r) 36554 +/- 17. Enzyme activity was dependent on the presence of Zn2+ ions and an exogenous thiol. Two molar equivalents of Zn2+ are bound/mol of subunit under reducing conditions. On exposure to the metal chelator EDTA, the two Zn2+ ions are removed, giving an inactive metal-depleted apo-ALAD. On oxidation of holo-ALAD, two disulphide bonds are formed with the loss of 1 mol of Zn2+/mol of subunit. The formation of the first disulphide led to the loss of catalytic activity. Replacement of the two bound Zn2+ ions with Co2+ resulted in the formation of a green protein with a spectrum indicative of the presence of charge-transfer bands from one or more cysteine-Co2+ ligands. While Mg2+ could not activate apo-ALAD alone, it was able to substitute for the second molar equivalent of bound Zn2+, leading to a further 4-fold stimulation in activity. The four cysteine residues involved in the formation of the two disulphide bonds were identified by protein-chemistry studies and were all located in a region of the protein extending from amino acid residues 120-134. Protein sequence data obtained in the present study has permitted the resolution of several differences between the published gene-derived protein sequences for ALAD from E. coli. |
| Links | |
| Keywords |
Amino Acid Sequence; Binding Sites; Cobalt/metabolism; Cobalt/pharmacology; Cysteine/analysis; Cysteine/metabolism; Disulfides/metabolism; Edetic Acid/pharmacology; Escherichia coli/enzymology; Hydrogen-Ion Concentration; Isoelectric Point; Kinetics; Magnesium/pharmacology; Metals/metabolism; Molecular Sequence Data; Nitrobenzoates/pharmacology; Oxidation-Reduction; Porphobilinogen Synthase/chemistry; Porphobilinogen Synthase/isolation & purification; Porphobilinogen Synthase/metabolism; Sulfhydryl Compounds/analysis; Zinc/metabolism; Zinc/pharmacology |
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