Bovine lens leucyl aminopeptidase (blLAP), a homohexameric metallopeptidase preferring bulky and hydrophobic amino acids at the N-terminus of (di)peptides, contains two Zn(2+) ions per subunit that are essential for catalytic activity. They may be replaced by other divalent cations with different exchange kinetics. The protein readily exchangeable site (site 1) can be occupied by Zn(2+), Mn(2+), Mg(2+), or Co(2+), while the tight binding site (site 2) can be occupied by Zn(2+) or Co(2+). We recently reported that introduction of Mn(2+) into site 1 generates a novel activity of blLAP toward CysGly [Cappiello, M., et al. (2004) Biochem. J. 378, 35-44], which in contrast is not hydrolyzed by the (Zn/Zn) enzyme. This finding, while disclosing a potential specific role for blLAP in glutathione metabolism, raised a question about the features required for molecules to be a substrate for the enzyme. To clarify the interaction of the enzyme with sulfhydryl-containing derivatives, (Zn/Zn)- and (Mn/Zn)blLAP forms were prepared and functional-structural studies were undertaken. Thus, a kinetic analysis of various compounds with both enzyme forms was performed; the crystal structure of (Zn/Zn)blLAP in complex with the peptidomimetic derivative Zofenoprilat was determined, and a modeling study on the CysGly-(Zn/Zn)blLAP complex was carried out. This combined approach provided insight into the interaction of blLAP with sulfhydryl-containing derivatives, showing that the metal exchange in site 1 modulates binding to these molecules that may result in enzyme substrates or inhibitors, depending on the nature of the metal.
|Autori interni:||CAPPIELLO, MARIO|
DEL CORSO, ANTONELLA
|Autori:||CAPPIELLO M; ALTERIO V; AMODEO P; DEL CORSO A; SCALONI A; PEDONE C; MOSCHINI R; DE DONATIS GM; DE SIMONE G; MURA U.|
|Titolo:||Metal Ion Substitution in the Catalytic Site Greatly Affects the Binding of Sulfhydryl-Containing Compounds to Leucyl Aminopeptidase|
|Anno del prodotto:||2006|
|Digital Object Identifier (DOI):||10.1016/j.jbbm.2006.01.009|
|Appare nelle tipologie:||1.1 Articolo in rivista|