MUTATIONAL ANALYSIS OF HUMAN UROPORPHYRINOGEN DECARBOXYLASE

Uroporphyrinogen decarboxylase (URO-D), a heme biosynthetic enzyme, ca talyzes the multi-step decarboxylation reaction converting uroporphyri nogen I or III to coproporphyrinogen I or III. The URO-D protein has b een purified from several sources and its gene has been cloned from ma ny organisms. In spite of this, little is known about the active site( s) of the enzyme. Inhibitor studies suggest that cysteine and histidin e residues are important for enzyme activity. We employed the Kunkel m ethod of site-directed mutagenesis to convert each of the six cysteine s in human URO-D to serine and each of the three conserved histidines to asparagine. Recombinant mutant URO-D's were expressed in Escherichi a coli, partially purified, and their kinetic properties compared to r ecombinant wild-type URO-D. All cysteine mutants retained approx. 40% wild-type enzyme activity, indicating that no single cysteine is absol utely critical for the integrity of the catalytic site. The three hist idine mutants also retained significant enzyme activity and one, (H339 N), displayed unique properties, The H339N mutation resulted in an enz yme with high residual activity but decarboxylation of intermediate re action products of the I isomer series was markedly abnormal. The hist idine at residue 339 is likely important in imparting isomer specifici ty.