Structure-Function relationship of Uroporphyrinogen decarboxylase (UroD)

MARTÍN GRAZIANO; DELFINA ROMERO; MARÍA DEL CARMEN RÍOS DE MOLINA

Rosario, Santa Fe (Argentina)

Congreso; XXLII Reunión anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2006

Uroporphyrinogen decarboxilase (UroD) is the fifth enzyme in the synthesis of haem.  It has been postulated that it is a homodimer in solution (Kd 0.1 μM), but the impact of dimerization in its activity is still not known.  The aim of this work is to study the equilibrium dimerization of UroD by means of Molecular Sieve Chromatography and Cross-Linking, and to characterize kinetically the dimer of UroD and its monomers using different substrates from the enzyme: Uroporphyrinogen III (Uro III) and Pentaporphyrinogen I (Penta I), to be able to determine whether the enzymatic activity depends on the dimerization state of the enzyme and whether exist differences regarding to the mechanism of action using the different substrates. UroD was expressed in a heterologous system, E. coli BL21, inducing the synthesis with IPTG 1 mM by 5 hs. The purification was made through a column of Ni2+ that retains in preferential form poliHis-UroD.  All the results confirm the hypothesis that the UroD presents a reversible equilibrium of dimerization. The Kd was determined by Gel Filtration and Cross-Linking obtaining a value of 0.27 ± 0.06 μM. Tests of enzymatic activity versus enzyme concentration were made in addition. With Penta I as substrate, the specific activity stays constant within the studied range of protein concentration, but however, when using Uro III as substrate, the specific activity increases with the enzymatic concentration. On the basis of this, UroD would behave differentially according to the substrate that is used: while the dimer presents a greater activity than monomer with Uro III as substrate, however with Penta I both form of the enzyme are equally active.