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 M(r) 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...

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Detalles Bibliográficos
Autores principales: Cardalda, C.A., Juknat, A.A., Princ, F.G., Batlle, A.
Formato: JOUR
Materias:
Cysteine residue(s)
Porphobilinogen deaminase
Porphobilinogen deaminase regulation
Protoporphyrin IX
Rat harderian gland
5,5' dithiobis(2 nitrobenzoic acid)
cysteine
n ethylmaleimide
porphobilinogen deaminase
protoporphyrin
uroporphyrin
animal tissue
article
catalysis
enzyme activity
enzyme analysis
enzyme kinetics
enzyme purification
enzyme substrate
Harder gland
male
molecular weight
nonhuman
priority journal
rat
Acceso en línea:http://hdl.handle.net/20.500.12110/paper_00039861_v347_n1_p69_Cardalda
Aporte de:
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires
Descripción
Sumario: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 M(r) 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, K(m) and V(m), calculated at 37°C and pH 8.0 were 1.1 μM and 170 pmol/min mg, respectively. The pH dependence of the apparent kinetic parameters revealed the ionization of residues with pK(A)/(ES) and pK(B)/(ES) of 7.4 ± 0.1 and 8.6 ± 0.1, respectively, and a pK(E) 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 μM 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.

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