Characterization of the two 5-aminolaevulinic acid binding sites, the A- and P-sites, of 5-aminolaevulinic acid dehydratase from Escherichia coli.
Experiments are described in which the individual properties of the two 5-aminolaevulinic acid (ALA) binding sites, the A-site and the P-site, of 5-aminolaevulinic acid dehydratase (ALAD) have been investigated. The ALA binding affinity at the A-site is greatly enhanced (at least 10-fold) on the binding of the catalytic metal ion (bound at the alpha-site). The nature of the catalytic metal ion, Mg2+ or Zn2+, also gave major variations in the substrate Km, P-site affinity for ALA, the effect of potassium and phosphate ions and the pH-dependence of substrate binding. Modification of the P-site by reaction of the enzyme-substrate Schiff base with NaBH4 and analysis of the reduced adduct by electro-spray mass spectrometry indicated a maximum of 1 mol of substrate incorporated/mol of subunit, correlating with a linear loss of enzyme activity. The reduced Schiff-base adduct was used to investigate substrate binding at the A-site by using rate-of-dialysis analysis. The affinity for ALA at the A-site of Mg alpha Zn beta ALAD was found to determine the Km for the reaction and was pH-dependent, with its affinity increasing from 1 mM at pH 6 to 70 microM at pH 8.5. The affinity of ALA at the P-site of Zn alpha An beta ALAD is proposed to limit the Km at pH values above 7, since the measured Kd for ALA at the A-site in 45 microM Tris, pH 8, was well below the observed Km (600 microM) under the same conditions. The amino group of the ALA molecule bound at the P-site was identified as a critical binding component for the A-site, explaining why ALA binding to ALAD is ordered, with the P-site ALA binding first. Structural requirements for ALA binding at the A- and P-sites have been identified: the P-site requires the carbonyl and carboxylate groups, whereas the A-site requires the amino, carbonyl and carboxylate groups of the substrate.
Spencer
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Department of Biochemistry, School of Biological Sciences, University of Southampton, U.K.
Jordan
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D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D000622:Aminolevulinic Acid / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism* / Q000648:ultrastructure*; D001665:Binding Sites; D001894:Borohydrides / Q000494:pharmacology; D002021:Buffers; D002250:Carbon Radioisotopes; D003956:Dialysis; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D000623:Porphobilinogen Synthase / Q000378:metabolism* / Q000648:ultrastructure*; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D001894:Borohydrides; D002021:Buffers; D002250:Carbon Radioisotopes; D008024:Ligands; D000622:Aminolevulinic Acid; D000623:Porphobilinogen Synthase; D008274:Magnesium; D015032:Zinc; D000409:Alanine