Effects on dimerization state and enzymatic activity of Uroporphyrinogen decarboxylase (UroD)

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

Rosario, Santa Fe (Argentina)

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

In order to determine the UroD Structure-Function relationship, we have evaluated the effect of diverse salts (KCl and (NH4)2 SO4), GdmHCl, and pH on UroD dimerization and the effects on its Enzymatic Activity. UroD has been expressed in E.coli BL21, induced with IPTG for 5 hours at 37°C. The purification was made through a Ni2+ column that retains in preferential form poliHis-UroD. We have studied the behavior of the enzyme using the technique of Cross-Linking and glutaraldehyde as the linking agent. These allow us to evaluate the degree of dimerization of UroD under different conditions. After completion of the technique, the effect of different conditions on the equilibrium dimerization of UroD was evaluated, and the results obtained indicate that the dimerization state can be altered by factors assayed. All the compounds orconditions studied produced a variation in the percentage of linked species (dimer). These results show that it is possible to alter the balance of UroD dimerization. These will allow us to evaluate the UroD enzymatic activity under different conditions, where it prevail one or another form of the enzyme, and to evaluate finally if the monomer of the UroD is active or not. According to this, we measured UroD activity varying the concentration of KCl, using Uro III as the substrate, andwe could verify that only the dimeric form would be active.In order to determine the UroD Structure-Function relationship, we have evaluated the effect of diverse salts (KCl and (NH4)2 SO4), GdmHCl, and pH on UroD dimerization and the effects on its Enzymatic Activity. UroD has been expressed in E. coli BL21, induced with IPTG for 5 hours at 37°C. The purification was made through a Ni2+ column that retains in preferential form poliHis-UroD. We have studied the behavior of the enzyme using the technique of Cross-Linking and glutaraldehyde as the linking agent. These allow us to evaluate the degree of dimerization of UroD under different conditions.