Mutational analysis of human uroporphyrinogen decarboxylase.
Uroporphyrinogen decarboxylase (URO-D), a heme biosynthetic enzyme, catalyzes the multi-step decarboxylation reaction converting uroporphyrinogen I or III to coproporphyrinogen I or III. The URO-D protein has been purified from several sources and its gene has been cloned from many organisms. In spite of this, little is known about the active site(s) of the enzyme. Inhibitor studies suggest that cysteine and histidine residues are important for enzyme activity. We employed the Kunkel method of site-directed mutagenesis to convert each of the six cysteines in human URO-D to serine and each of the three conserved histidines to asparagine. Recombinant mutant URO-D's were expressed in Escherichia coli, partially purified, and their kinetic properties compared to recombinant wild-type URO-D. All cysteine mutants retained approx. 40% wild-type enzyme activity, indicating that no single cysteine is absolutely critical for the integrity of the catalytic site. The three histidine mutants also retained significant enzyme activity and one, (H339N), displayed unique properties. The H339N mutation resulted in an enzyme with high residual activity but decarboxylation of intermediate reaction products of the I isomer series was markedly abnormal. The histidine at residue 339 is likely important in imparting isomer specificity.
Wyckoff
E E
EE
Department of Medicine University of Utah, School of Medicine, Salt Lake City 84132, USA.
Phillips
J D
JD
Sowa
A M
AM
Franklin
M R
MR
Kushner
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D005033:Ethylmaleimide / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014575:Uroporphyrinogen Decarboxylase / Q000737:chemistry / Q000235:genetics*
D011994:Recombinant Proteins; D006639:Histidine; D014575:Uroporphyrinogen Decarboxylase; D003545:Cysteine; D005033:Ethylmaleimide