New role for leucyl aminopeptidase in glutathione turnover

Abstract

A manganese-dependent cysteinyl-glycine hydrolysing activity has been purified to electrophoretic homogeneity from bovine lens. The characterization of the purified enzyme (molecular mass of the native protein, molecular mass of the subunit and extensive primary structure analysis) allowed the unequivocal attribution of the cysteinyl-glycine hydrolysing activity, which is usually associated with alanyl aminopeptidase (EC 3.4.11.2) or membrane-bound dipeptidase (EC 3.4.13.19), to LAP (leucyl aminopeptidase; EC 3.4.11.1). Analysis of the pH dependence of Cys-Gly hydrolysis catalysed by LAP, supported by a molecular modelling approach to the enzyme-substrate conformation, gave insights into the ability of the enzyme to recognize Cys-Gly as a substrate. Due to the effectiveness of LAP in hydrolysing Cys-Gly (K(m)=0.57 mM, kcat=6.0x10(3) min(-1) at pH 7.4 and 25 degrees C) with respect to other dipeptide substrates, a new role for this enzyme in glutathione turnover is proposed.

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

“…Another distinctive feature common to peptidases of the M17 family, which is exhibited by the Cys-Gly hydrolase from Patella is its remarkable sensitivity to bestatin, able to inhibit the enzyme with an IC 50 in the nM range. Moreover, the pH dependence of the kinetic parameters of the purified enzyme is consistent with the proposed mechanism of action of M17 family peptidases, that, as generally accepted 25,31 , can catalyze peptide hydrolysis only when the a-aminogroup of the substrate is unprotonated. All these features and the mass spectrometry characterization of the purified enzyme lead to the conclusion that also in mollusks, Cys-Gly hydrolase activity is associated with a protein of the peptidase family M17.…”

“…Cys-Gly hydrolase activity was assayed as previously described 25 , by a colorimetric method based on the reaction between cysteine produced in the hydrolysis of Cys-Gly and ninhydrin. Ninhydrin specifically reacts with cysteine in the presence of a mixture of acetic acid and hydrochloric acid, forming a purple complex with a maximum of absorbance at 560 nm.…”

“…Cys-Gly hydrolase activity has been associated with the brush border aminopeptidase M (alanyl aminopeptidase, EC 3.4.11.2) or the membrane-bound dipeptidase (EC 3.4.13.19) 22,23 . However, a growing body of evidence, mainly obtained for rat liver 24 and bovine lens 25 , has recently led to the association of Cys-Gly hydrolase activity in mammals with leucyl aminopeptidase (E.C 3.4.11.1), a metallopeptidase that belongs to the MEROPS peptidase family M17. While enzymes able to hydrolyze CysGly have been characterized in vertebrates and microorganisms 26 , to our knowledge no information is available on Cys-Gly hydrolase activities in mollusks.…”

Hydrolysis of S-aryl-cysteinylglycine conjugates catalyzed by porcine kidney cortex membrane dipeptidase

“…3.4.13.18) (Kaur et al, 2009;Teufel et al, 2003), leucyl aminopeptidase (also known as cytosol aminopeptidase; peptidase S; E.C. 3.4.11.1) (Cappiello et al, 2004;Jösch et al, 1998;Jösch et al, 2003), and prolyl aminopeptidase (also known as cytosol aminopeptidase V; E.C. 3.4.11.5) (Turzynski and Mentlein, 1990).…”

Unusual activity pattern of leucine aminopeptidase inhibitors based on phosphorus containing derivatives of methionine and norleucine

“…In mammals, the predominant function of LAP is considered to be its involvement in the maturation, activation, modulation and degradation of bioactive peptides [1,2,[5][6][7]. Recently, the leucine aminopeptidase has been proposed to be the key enzyme responsible for glutathione turnover in the liver, and thus influences the redox status of the cell [8][9][10]. The processing of antigenic peptides by LAP has a minor role in epitope generation for presentation by the major histocompatibility complex class I molecules [11,12].…”