Rat kidney porphobilinogen deaminase kinetics. Detection of enzyme-substrate complexes.
OBJECTIVE
Acute intermittent porphyria (AIP) is an inherited disease resulting from a reduced activity of the enzyme porphobilinogen deaminase (PBG-D). The kidney is an important target for numerous porphyrinogenic drugs and it may contribute to the clinical manifestations of porphyric attacks. An evaluation of kidney PBG-D role in the AIP pathophysiology requires detailed information on kidney PBG-D properties, under normal conditions.


METHODS
Rat kidney PBG-D was purified to homogeneity and initial reaction velocities were calculated by measuring uroporphyrinogen I formation at pH 8.2 for different incubation times (0-20 min) and over a wide range of substrate concentrations (0.8-66 microM).


RESULTS
Purified rat kidney PBG-D is a monomeric enzyme showing only a single protein band after SDS-PAGE, Western blot and isoelectric focusing (pI 4.9). Its molecular mass is 40 +/- 2.3 kDa, determined by SDS-PAGE and 39.8 +/- 2 kDa by gel filtration chromatography. Rat kidney PBG-D has an unusual kinetic behaviour, exhibiting a deviation from the Michaelis-Menten hyperbola. PBG-D kinetic data required a fitting to an equation of higher degree, leading to the following apparent kinetic constants: K(1) = 2.08 +/- 0.01 microM and K(2) = 0.102 +/- 0.003 microM.


CONCLUSIONS
The values of these constants fulfil the restriction 4K(2) < or = K(1)(2), necessary for the occurrence of isoenzymes, interpreted in this work as enzyme-substrate intermediates. The initial reaction velocity expression here defined, correlates with an enzyme carrying only one active site but allowing, through conformational changes, the detection of at least two enzyme-substrate intermediates formed during PBG-D reaction.
Noriega
Guillermo
G
Centro de Investigaciones sobre Porfirinas y Porfirias, CONICET, Departamento de Química Biológica, 1428 Buenos Aires, Argentina.
Mattei
Guillermo
G
Batlle
Alcira
A
Juknat
Adela Ana
AA
D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D011163:Hydroxymethylbilane Synthase / Q000378:metabolism*; D007525:Isoelectric Focusing; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D051381:Rats; D013379:Substrate Specificity
D011163:Hydroxymethylbilane Synthase