5-Aminolaevulinic acid dehydratase: characterization of the alpha and beta metal-binding sites of the Escherichia coli enzyme.

Abstract

The alpha and beta metal-binding sites of 5-aminolaevulinic acid dehydratase (ALAD) (porphobilinogen synthase, EC 4.2.1.24) from Escherichia coli were investigated to determine the function of each metal ion and the role of the reactive cysteines in metal binding. Occupancy of the alpha site by Zn2+ restored virtually all catalytic activity to the inactive metal-depleted ALAD (apoALAD). Occupancy of the alpha site by Co2+ also yielded an active enzyme and resulted in a charge-transfer band indicative of a single cysteine amongst the metal ligands. Subsequent labelling of this cysteine residue with 14C-labelled N-ethylmaleimide, followed by peptide analysis, indicated the involvement of Cys-130. The metal ion at the alpha site is thought to be essential for binding of the second molecule of substrate at the A substrate-binding site that forms the acetic acid side of the product, porphobilinogen. Binding of Zn2+ to the beta site restored little activity if the alpha site was unfilled. Metal ion binding to the beta site could be monitored by following the change in protein fluorescence with Zn2+ titration of apoALAD at pH 6. A conformational change upon beta site occupancy may explain why binding of Mg2+ at the alpha site can occur only if Zn2+ is bound at the beta site. The binding of Co2+ at the beta site produced an inactive enzyme that exhibited a charge-transfer band indicative of at least three cysteine ligands.