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 at 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 Ki 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.