Beef-Liver 5-Aminolevulinic Acid Dehydratase.

Beef liver 5-aminolevulinate dehydratase was purified 700-fold by salt fractionation, heat treatment, gel filtration and ion-exchange chromatography. The final product was homogeneous by electrophoretic, ultracentrifugal and immunological criteria and had a molecular weight, by the low-speed equilibrium method, of 260000. Treatment with sodium dodecylsutfate and 2-mercaptoethanol dissociated the enzyme into dimeric subunits which could be further dissociated into monomeric subunits by treatment with 7.3 M guanidine hydrochloride and dithioerythreitol. The molecular weight of the monomeric subunit and the results of amino acid analysis suggested the presence of 14 subunits per mole. Carboxymethylation and hydrolysis showed the presence of 56 cysteine residues per mole, approximately half of which were available for titration with p-ehloromereuribenzoate in the urea-denatured enzyme. The enzyme has a pH optimum of 6.3 and a K_m of 0.46 mM in the crude state and 0.15 mM when pure. Thiol activation was required for catalytic activity and some evidence was obtained for a requirement for zinc as a co-factor. Heavy metals inhibited the enzyme in a complex manner. Zinc acted as a competitive inhibitor, lead acted as a non-competitive inhibitor, while cadmium acted in a unique manner, causing inhibition at low concentrations of subtrate and stimulation at high levels of substrate. [ABSTRACT FROM AUTHOR]