Purification and characterization of 5-aminolaevulinic acid dehydratase from Escherichia coli and a study of the reactive thiols at the metal-binding domain.
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.
Spencer
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School of Biological Sciences, Queen Mary and Westfield College, University of London, U.K.
Jordan
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PM
D000595:Amino Acid Sequence; D001665:Binding Sites; D003035:Cobalt / Q000378:metabolism / Q000494:pharmacology; D003545:Cysteine / Q000032:analysis / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D009579:Nitrobenzoates / Q000494:pharmacology; D010084:Oxidation-Reduction; D000623:Porphobilinogen Synthase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000032:analysis*; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
D004220:Disulfides; D008670:Metals; D009579:Nitrobenzoates; D013438:Sulfhydryl Compounds; D003035:Cobalt; D004492:Edetic Acid; D000623:Porphobilinogen Synthase; D008274:Magnesium; D015032:Zinc; D003545:Cysteine