Metal Ion Substitution in the Catalytic Site Greatly Affects the Binding of Sulfhydryl-Containing Compounds to Leucyl Aminopeptidase^,

Abstract

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.

A 52-kDa Leucyl Aminopeptidase from Treponema denticola Is a Cysteinylglycinase That Mediates the Second Step of Glutathione Metabolism

“…CGA, purified recombinant T. denticola 52-kDa protein; GGT, purified T. denticola ␥-glutamyltransferase (19); Cysta, T. denticola cystalysin (L-cysteine desulfhydrase) (18). (46,47) identified the Cys-Gly hydrolyzing activity in bovine lens and showed that the protein previously characterized as an M17 leucine aminopeptidase was the enzyme involved. They compared the kinetics of their protein with Cys-Gly versus Leu-Gly and found that Cys-Gly was the preferred substrate, at least with Mn 2ϩ as the cation.…”

Purification and characterization of a Cys-Gly hydrolase from the gastropod mollusk,Patella caerulea

“…Since it has been demonstrated that the activity of peptidases belonging to family M17 varies depending on the metal ions present in the active site, the enzymes of this family have been adopted as model enzymes to clarify the mechanisms of metal ion-dependent enzymatic catalysis [32][33][34] . Our purification of the enzyme from Patella is an extremely fast procedure.…”

Hydrolysis of Amides

“…Recently, it has been found that exchange of Zn 2 þ in site M1 against Mn 2 þ can even lead to a totally new enzymatic activity. Whereas the dipeptide CysGly cannot be hydrolyzed by Zn/Zn blLAP, and actually acts as a competitive inhibitor for the hydrolysis of LeuGly, the Mn/Zn enzyme readily converts this cysteinyl containing substrate [242,243]. Interestingly, the metal binding site M1 in ppLAP that has been heterologously produced in E. coli is already occupied by Mn 2 þ , which leads to a much more active enzyme than when this site contains Zn 2 þ (ID 1313).…”