Mechanistic studies on enzyme-catalysed dimerization reactions

Abstract

5-Aminolevulinic acid dehydratase catalyses a dimerization reaction
whereby the two identical substrate molecules, 5-aminolevulinic acid,
are condensed together to yield porphobilinogen, the monopyrrolic precursor
of porphyrin biosynthesis. A new procedure for the purification of the
enzyme from human erythrocytes was developed and yielded 27.5 mg of pure
enzyme protein in good recovery with a high specific activity (24.0 units/mg protein). The human enzyme was treated with [4-14c] aminolevulinic acid and the resulting Schiff base complex was reduced with sodium borohydride. The modified protein was cleaved with cyanogen bromide and the peptide containing the label was isolated and sequenced. Similar studied were performed on the enzyme isolated from bovine liver. Comparison of the two active site sequences revealed a common hexapeptide which had the following structure where X* is amino acid -4-14C 5-aminolevulinic acid adduct (probably ALA-lysine).

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