Study of kinetic behavior of convertases

Abstract

The protein-convertases (PCs) are proteolytic enzymes that belong to the family of subtilisin-like serine proteases. These enzymes are found in the secretory pathways and in secretory vesicles of trans-Golgi in the eukaryotic cell and they are involved in the processing of pro-proteins, pro-hormones, receptors, and pro-viral capsid proteins. Convertases are Ca2+-dependent endopeptidases that recognize and cleave basic sequence Lys-Arg or Arg-Arg present at the C-terminal domain of its protein substrates, and these enzymes are involved in several pathophysiological processes such as: processing of tissue growth factors TGF-² and BMP-4, pro-insulin receptor, glycoprotein gp160 of the HIV virus, the glycoprotein of Ebola virus and anthrax protective antigen. The mammalian PCs family comprises seven members, furin, PACE4, PC4, PC5/6, PC7, PC1 and PC2. The convertase Kex2 is considered the archetype of PCs and was first identified in Saccharomyces cerevisiae. In this project we aimed to study the specificity of convertases Kex2, PC1 and Furin on the hydrolysis of fluorescent type FRET peptide substrates, as well as determine the individual rate constants of each catalytic step of convertases, in other words, determining the binding step of substrate (association constant k1 and dissociation k-1) and catalysis of the substrate (acylation constant k2 and hydrolysis k3) using monitoring techniques of the hydrolysis of the fluorescent substrates, by stopped-flow, and by the effect of temperature on the different rate constants in the enzyme reaction. Also will be studied the effect of allosteric modulators on the activity of convertases, such as: the binding of mono- and divalent metals to enzymes and the effect of interaction with glycosaminoglycans. The effect of allosteric modulation of ligands will also be assessed by the conformational change of the enzymes by intrinsic fluorescence and circular dichroism spectroscopy. Thus, the set of information derived from studies of specificity, thermodynamic analysis, and performance of modulators will allow better understanding of the kinetic behavior of these proteolytic enzymes. (AU)