Investigation of the nature of the two metal-binding sites in 5-aminolaevulinic acid dehydratase from Escherichia coli.
Two distinct metal-binding sites, termed alpha and beta, have been characterized in 5-aminolaevulinic acid dehydratase from Escherichia coli. The alpha-site binds a Zn2+ ion that is essential for catalytic activity. This site can also utilize other metal ions able to function as a Lewis acid in the reaction mechanism, such as Mg2+ or Co2+. The beta-site is exclusively a transition-metal-ion-binding site thought to be involved in protein conformation, although a metal bound at this site only appears to be essential for activity if Mg2+ is to be bound at the alpha-site. The alpha- and beta-sites may be distinguished from one another by their different abilities to bind divalent-metal ions at different pH values. The occupancy of the beta-site with Zn2+ results in a decrease of protein fluorescence at pH 6. Occupancy of the alpha- and beta-sites with Co2+ results in u.v.-visible spectral changes. Spectroscopic studies with Co2+ have tentatively identified three cysteine residues at the beta-site and one at the alpha-site. Reaction with N-ethyl[14C]maleimide preferentially labels cysteine-130 at the alpha-site when Co2+ occupies the beta-site.
Spencer
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Department of Biochemistry, Southampton University, U.K.
Jordan
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PM
D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003035:Cobalt / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D000623:Porphobilinogen Synthase / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013054:Spectrophotometry, Atomic; D015032:Zinc / Q000378:metabolism
D008670:Metals; D003035:Cobalt; D004228:Dithionitrobenzoic Acid; D000623:Porphobilinogen Synthase; D008274:Magnesium; D015032:Zinc; D003545:Cysteine; D005033:Ethylmaleimide