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.
Cardalda
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Departamento de Quimica Biologica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires Ciudad Universitaria, 1428 Buenos Aires, Argentina.
Juknat
A A
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Princ
F G
FG
Batlle
A
A
D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005033:Ethylmaleimide / Q000494:pharmacology; D006243:Harderian Gland / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D011163:Hydroxymethylbilane Synthase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D011166:Porphyrins / Q000032:analysis / Q000302:isolation & purification; D011524:Protoporphyrins / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014577:Uroporphyrinogens / Q000096:biosynthesis
D004791:Enzyme Inhibitors; D011166:Porphyrins; D011524:Protoporphyrins; D013438:Sulfhydryl Compounds; D014577:Uroporphyrinogens; C028025:protoporphyrin IX; D011163:Hydroxymethylbilane Synthase; D005033:Ethylmaleimide