Comparative studies on the 5-aminolaevulinic acid dehydratases from Pisum sativum, Escherichia coli and Saccharomyces cerevisiae

5-Aminolaevulinic acid dehydratase (ALAD) is an essential enzyme in most organisms, catalysing an inaugural step in the tetrapyrrole biosynthetic pathway, the Knorr-type condensation reaction of two molecules of 5-aminolaevulinic acid (ALA) to form the monopyrrole porphobilinogen. ALADs can be conveniently separated into two main groups: those requiring Zn²⁺ for activity (typified here by the enzymes from Escherichia call and Saccharomyces cerevisiae, yeast) and those requiring Mg²⁺ (represented here by the enzyme from Pisum sativum, pea). Here we describe a detailed comparison of these two metal-dependent systems. Kinetically influential ionizations were identified by using pH-dependent kinetics. Groups with pK_a values of approx. 7 and 10 (assigned to cysteine and lysine residues) were detected in the free enzyme and enzyme-substrate states of all three enzymes, and a further ionizable group with a pK_a of approx. 8.5 (assigned to histidine) was found to be additionally important to the yeast enzyme. The importance of these residues was confirmed by using protein modifying reagents. Shifts in the pK_a values of the pea and E. coli enzymes consequent on E-S complex formation suggest a change to a less hydrophobic microenvironment when substrate binds. Studies with inhibitors revealed that the three enzymes exhibit differential susceptibilities and, in the case of succinylacetone, this is reflected in K_i values that vary by three orders of magnitude. In addition, the crystallization of the yeast ALAD is described, raising the possibility of an X-ray-derived three-dimensional structure of this enzyme.