Characterization of the multiple forms of hydroxymethylbilane synthase from rat spleen

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Abstract
Phenylhydrazine treatment induced hydroxymethylbilane synthase activity in rat spleen, erythrocytes and liver by 40-fold, 7.5-fold and 6-fold, respectively. Five multiple forms of the enzyme were resolved by DEAE-cellulose chromatography. In the presence of phenylmethanesulfonyl fluoride only 3 forms, 2 major and 1 minor, were resolved by the fractionation, suggesting that 2 of the original forms arose by proteolytic modification. Heat treatment (70.degree. C) in the presence of proteinase inhibitor converted one of the major forms into the other major form. Product isomer analysis suggested that this heat-labile form represented an enzyme-substrate covalent intermediate and not a hydroxymethylbilane synthase-uroporphyrinogen III synthase complex. Identical elution profiles and kinetic properties of the enzymes form rat spleen and erythrocytes suggested that the enyzme isolated from spleen was possibly from stored erythrocytes. Sephadex G-75 chromatography of the heat-stable DEAE-cellulose-purified form yielded pure enzyme as judged by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis. The MW was 43,000 .+-. 1500. Initial-velocity studies on all enzyme forms showed a hyperbolic dependence of velocity on substrate concentration, demonstrating the existence of displacement-type mechanism. For the heat-stable form Vmax varied with pH as a typical bell-shaped curve, indicating that 2 ionizable groups with pK values of 7.4 and 8.8 are important for catalysis. Km decreased with decreasing pH on the acid side of the pH optimum, suggesting the absence of ionization of a group with pK 7.4 in free enzyme or substrate.