Characterization of a recombinant pea 5-aminolevulinic acid dehydratase and comparative inhibition studies with the Escherichia coli dehydratase

Pea 5-aminolevulinic acid dehydratase (ALAD) was purified 200-fold from a recombinant overproducing strain of Escherichia coli, yielding an octameric enzyme with a specific activity of 280 units mg(-1) Divalent metal ions were essential, Mg2+, Mn2+, and Co2+ ions all supporting activity, whereas Zn2+ ions could not. Equilibrium dialysis and atomic absorption studies revealed two Mg2+ ion binding sites per subunit. Pea ALAD bound the substrate 5-aminolevulinic acid covalently through a Schiff base al the P-site, electrospray mass spectrometry of the reduced enzyme-ALA Schiff base complex showing the presence of one P-site per subunit. The amino acid residue modified by ALA was identified by MALDI-MS and Edman sequencing as Lys-293, analogous to the active site Lys-247 of E. coli ALAD and Lys-252 of mammalian ALAD. Comparative studies of pea ALAD with E. coli ALAD using the inhibitors 3-acetyl-4-oxoheptane-1,7-dioic acid (AOHD) and succinylacetone (SA) indicated similar modes of inhibition, with the formation of a Schiff base complex between the inhibitors and the active site lysine. Studies with the ALA homolog, 4-amino-3-oxobutanoic acid (AOB), revealed that it is specific for the A-site of both the pea and E. coli ALADs. An interesting difference exists between the enzymes, however, pea ALAD being far more susceptible to inhibition with AOB than the E coli enzyme. AOB bound 10 times better to the A-site of pea ALAD compared to the substrate,ALA. Despite the 2000 times lower K-1 of AOB for pea ALAD, no abortive Schiff base intermediate, between enzyme-bound ALA at the P-site and AOB bound at the A-site, could be demonstrated.