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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todo:paper_00039861_v347_n1_p69_Cardalda2023-10-03T13:57:01Z Rat harderian gland porphobilinogen deaminase: Characterization studies and regulatory action of protoporphyrin IX Cardalda, C.A. Juknat, A.A. Princ, F.G. Batlle, A. 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 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. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_00039861_v347_n1_p69_Cardalda |
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Universidad de Buenos Aires |
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I-28 |
| repository_str |
R-134 |
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Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
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 |
| spellingShingle |
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 Cardalda, C.A. Juknat, A.A. Princ, F.G. Batlle, A. Rat harderian gland porphobilinogen deaminase: Characterization studies and regulatory action of protoporphyrin IX |
| topic_facet |
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 |
| description |
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. |
| format |
JOUR |
| author |
Cardalda, C.A. Juknat, A.A. Princ, F.G. Batlle, A. |
| author_facet |
Cardalda, C.A. Juknat, A.A. Princ, F.G. Batlle, A. |
| author_sort |
Cardalda, C.A. |
| title |
Rat harderian gland porphobilinogen deaminase: Characterization studies and regulatory action of protoporphyrin IX |
| title_short |
Rat harderian gland porphobilinogen deaminase: Characterization studies and regulatory action of protoporphyrin IX |
| title_full |
Rat harderian gland porphobilinogen deaminase: Characterization studies and regulatory action of protoporphyrin IX |
| title_fullStr |
Rat harderian gland porphobilinogen deaminase: Characterization studies and regulatory action of protoporphyrin IX |
| title_full_unstemmed |
Rat harderian gland porphobilinogen deaminase: Characterization studies and regulatory action of protoporphyrin IX |
| title_sort |
rat harderian gland porphobilinogen deaminase: characterization studies and regulatory action of protoporphyrin ix |
| url |
http://hdl.handle.net/20.500.12110/paper_00039861_v347_n1_p69_Cardalda |
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