Sequence and structure of the rat housekeeping PBG-D isoform.

Porphobilinogen deaminase (PBG-D), a key enzyme in the tetrapyrrole biosynthetic pathway, is encoded by a single gene containing two different promoters. The upstream promoter, found in all cell types, initiates the transcription of the housekeeping PBG-D isoform, whereas the downstream one is erythroid-specific. In this study, we provide the first full sequence of a 1086bp cDNA covering the coding region for the rat ubiquitous PBG-D and its primary amino acid sequence. The cDNA encodes a 39,361 Da protein composed of 361 amino acids. Nucleotide sequence comparison between both isoforms from rat shows similarities of 99.5%, with four changes (C/G) in exon 8 and only one (C/A) in exon 12. Secondary structure prediction reveals that 76.5% of the amino acids from exon 1 are located in a loop. Potential phosphorylation, glycosylation, and myristoylation sites were revealed through motif searches. Housekeeping PBG-D contains coiled-coil segments known to be involved in dynamic rearrangements in the active site.

Rat harderian gland porphobilinogen deaminase: characterization studies and regulatory action of protoporphyrin IX.

Properties of purified porphobilinogen deaminase (PBG-D; EC 4.3.1.8) from rat harderian gland are here presented. The enzyme behaves as a monomer of Mr 38 +/- 2 kDa and is optimally active at pH 8.0-8.2. Its activation energy, determined by an Arrhenius plot, is 76.1 kJ/mol. Initial velocity studies showed a linear progress curve for uroporphyringen I formation and a hyperbolic dependence of the initial rate on substrate concentration, indicating the existence of a sequential displacement mechanism. Apparent kinetic constants, Km and Vm, calculated at 37 degrees C and pH 8.0 were 1.1 microM and 170 pmol/min mg, respectively. The pH dependence of the apparent kinetic parameters revealed the ionization of residues with pKAES and pKBES of 7.4 +/- 0.1 and 8.6 +/- 0.1, respectively, and a pKE value of 8.0 +/- 0.1. Incubation of PBG-D with 5.0 mM N-ethylmaleimide and 5.0 mM 5,5'-dithiobis(2-nitrobenzoic acid) at pH 8.0 led to inhibitions of 70 and 50%, respectively. The effect of pH, as well as the effect of thiol reagents, on enzyme activity strongly suggests the involvement of cysteine residue(s) in the mechanism of uroporphyrinogen I biosynthesis, in both the catalytic reaction and the substrate binding. Rat harderian gland PBG-D activity decreased with increasing concentrations of protoporphyrin IX, reaching a 40% inhibition at the in vivo concentration of the porphyrin and 7 microM PBG. Even at saturating concentrations of substrate, inhibition by protoporphyrin was not completely reversed. So, accumulated porphyrin may act as an regulator of PBG-D activity in rat harderian gland.