Effect of hemeproteins on the protease Thimet- Oligopeptidase
1Ferreira, J.C., 2Castro, L.M., 3Machado, M.F.M., 3Oliveira, V., 2Ferro, E.S., 4Juliano, M.A. e 1Nantes, I.L
1Centro Interdisciplinar de Investigação de Bioquímica da Universidade de Mogi das Cruzes (UMC); 2Instituto de Ciências Biológicas da Universidade de São Paulo (USP); 3Neurociências da Universidade da Cidade de São Paulo (UNICID); 4Departamento de Biofísica da Universidade Federal de São Paulo (UNIFESP)
The endopeptidase EC.3.4.24.15, also known as timet-oligopeptidase (TOP), is a metallopeptidase. This enzyme contains Zn2+ in the active site that participates in the catalytic process. EP24.15 contains fourteen cysteine residues and is activated by DTT. In order to determine the specific role these cysteine residues in activation process of the enzyme, we produced mutant forms in which specific cysteine residues were replaced by serine residues (C246S, C248S, C248S/C253S, C246S/C248S, C246S/C253S and C246S/C248S/C253S). The comparative activity of the mutant and the wild type enzymes all of them activated by DTT and hemoglobin (Hb), myoglobin, apocytochrome c, Fe (III) and Fe(II) cytochrome c (cytc) and free base cytochrome c (cytc) was determined by fluorimetric assays in the presence of fluorescent substrates. The hemeproteins were used as a model for a possible interaction of TOP with cytoglobins, neuroglobins or even cytochrome c during the apoptotic process. DTT promoted the activation of wild type TOP and of the mutant C246S/C248S. Contrary to DTT, cytochrome c Fe(III) was efficient to activate C248S, suggesting that the mutation at the 248 position favored the activation process. In fact, circular dichroism analyis of C248S in the presence of cytc revealed structural alterations probable due to interactions between the proteins. Otherwise, in the reduced form (FeII), cytc inhibited all TOP forms. Hemoglobin, a hemeprotein with quaternary structure promoted significant activation of the mutant C246S/C248S but was inefficient to activate double mutants with changes at the 253 position. This result suggests that the activation process involves interactions with the cysteine 253. However, the mutant C246S/C248S/C253S exhibited low degree activation in the presence of hemoglobin, probably due to specific structural alterations promoted by this type of mutation. These results show that the activation of TOPs by hemeproteins are extremely specific and complex processes and requires more detailed investigations. Supported by FAPESP, CNPq and FAEP-UMC.
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