Caracterização de uma aspartil peptidase do fluido pericárdico humano utilizando peptídeos fluorescentes em torno da sequência do sítio reativo da calistatina

Abstract

Despite having structural similarities, sequencial homologies and a common catalytic mechanism for the hydrolysis of their substrates, aspartyl peptidases are enzymes that have very different preferences regarding interactions between amino acid residues at the catalytic site. These differences are important from a biological point of view, because each tissue/cell has its own metabolic conditions and needs, according to the chemical/biochemical components available in the medium, such as substrates, cofactors, inhibitors and products. In this study, an aspartyl peptidase from human pericardial fluid was purified through chromatographic steps and the kinetic parameters were determined according to each synthetic substrate modified at the reactive site of callistatin. The isolated enzyme had an apparent molecular mass of 141 kDa and the aspartyl peptidase activity was observed only in this molecular mass region. The data obtained from enzymatic kinetics showed us that the change in the position of the P1' amino acid significantly influenced its activity. We used these data for a comparative study with observed data for other human aspartic peptidases isolated from other sources using the same set of substrates obtained under the same assay conditions and the aspartyl peptidase’s pericardial isoform showed greater catalytic similarity with hepatic cathepsin D when compared to enzyme isolated from spleen. The calistatin reactive site sequences’ hydrolysis by aspartyl peptidase from human pericardial fluid suggests that the enzyme may interfere with physiological processes in which calistatin is involved.