STRUCTURE-FUNCTION RELATIONSHIP OF UROPORPHYRINOGEN DECARBOXYLASE (UROD)

GRAZIANO, M; ROMERO, D.M; RÍOS DE MOLINA, M.C

Rosario, Santa Fe

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2006

SAIB, Sociedad Argentina de Investigación Bioquímica y Biología Molecular

The aim of this work is to study the UroD dimerization equilibrium, and characterize kinetically the dimeric as well as the monomeric form of UroD. This will allow us to determine if the enzymatic activity is dependent on the oligomerization state of the enzyme and if there are differences in the action mechanism, with different substrates. 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 poliHis-UroD. By Gel Filtration and Cross-Linking, all the results obtained confirm the hypothesis that UroD has a reversible dimerization equilibrium. We have obtained a Kd of 0,27 ± 0,06 ìM by Gel Filtration. Tests of Enzymatic activity vs Enzyme concentration were made in addition. Specific activity with Pentagen I remain constant within the range of protein concentration studied; however, using Urogen III as substrate, the specific activity increases with the enzymatic concentration in the same protein range. Then, UroD would behave in a different way according to the substrate that is used: with Urogen III the dimer displays a greater activity than monomer, but with Pentagen I, both of them are equally active. Other tests are being made to verify the hypotheses raised by this work, using diverse methodologies to measure the activity of UroD affecting its dimerization state (effect of salts, pH, etc).