Glycosylasparaginase-catalyzed Synthesis and Hydrolysis of β-Aspartyl Peptides

Abstract

␤-Aspartyl di-and tripeptides are common constituents of mammalian metabolism, but their formation and catabolism are not fully understood. In this study we provide evidence that glycosylasparaginase (aspartylglucosaminidase), an N-terminal nucleophile hydrolase involved in the hydrolysis of the N-glycosidic bond in glycoproteins, catalyzes the hydrolysis of ␤-aspartyl peptides to form L-aspartic acid and amino acids or peptides. The enzyme also effectively catalyzes the synthesis of ␤-aspartyl peptides by transferring the ␤-aspartyl moiety from other ␤-aspartyl peptides or ␤-aspartylglycosylamine to a variety of amino acids and peptides. Furthermore, the enzyme can use L-asparagine as the ␤-aspartyl donor in the formation of ␤-aspartyl peptides. The data show that synthesis and degradation of ␤-aspartyl peptides are new, significant functions of glycosylasparaginase and suggest that the enzyme could have an important role in the metabolism of ␤-aspartyl peptides.␤-Aspartyl and ␥-glutamyl peptides are normal constituents of mammalian urine (1, 2) and tissues (3). Although ␥-glutamyltransferase (EC 2.3.2.2, GGT) (1) 1 is the key enzyme in the synthesis and hydrolysis of ␥-glutamyl compounds such as glutathione in the ␥-glutamyl cycle (4), little is known about the metabolism of ␤-aspartyl peptides.Glycosylasparaginase (GA; aspartylglucosaminidase;asparagine; ␤-aspartylglucosamine; GlcNAc-Asn) during degradation of glycoproteins. Genetic deficiency of glycosylasparaginase causes a lysosomal storage disease aspartylglycosaminuria (McKusick 208400) that is the most common disorder of glycoprotein degradation in humans and is clinically characterized by severe mental and motor retardation (5, 6).Glycosylasparaginase is a member of the recently described structural superfamily of enzymes termed as N-terminal nucleophile (Ntn) hydrolases (7). The hydrolysis of ␤-aspartylglycosylamines catalyzed by glycosylasparaginase is initiated by the binding of the ␤-aspartyl moiety into the active site of the enzyme through its free ␣-amino and ␣-carboxyl groups (8, 9). The enzyme uses the ␥-hydroxyl and ␣-amino group of its ␤-chain N-terminal threonine as an active site nucleophile and general base in the formation of ␤-aspartyl enzyme, which is subsequently deacylated by water to L-aspartic acid (10, 11). The GA-catalyzed hydrolysis of L-asparagine occurs in a similar manner, resulting in the formation of ␤-aspartyl enzyme and ammonia (12). The mechanism of action of glycosylasparaginase and the structural properties of its substrate-binding site (13) led us to consider that the enzyme might have a role in the metabolism of ␤-aspartyl peptides. In the present study, we demonstrate that synthesis and degradation of ␤-aspartyl peptides are new significant functions of glycosylasparaginase. This suggests that glycosylasparaginase could have an important role in the metabolism of ␤-aspartyl peptides present in body fluids and tissues. EXPERIMENTAL PROCEDURESMaterials-GA was purified to homogeneity from an NIH-3T3 cell line overexpre...

The Human Asparaginase-like Protein 1 hASRGL1 Is an Ntn Hydrolase with β-Aspartyl Peptidase Activity

“…The human lysosomal AGA is the only other known mammalian enzyme for which β-aspartyl peptidase activity had been previously observed (32), however; the reported k cat /K M values of AGA for a variety of such substrates are much lower relative to those for hASRGL1.…”

Depletion of L-asparagine supply and apoptosis of leukemia cells induced by human glycosylasparaginase

“…4 Lymphoblasts were continuously grown in an RPMI-1640 (HyClone, Logan, Utah, USA) medium supplemented with 15% of fetal calf serum (BioClear, Wiltshire, UK), 2 mM L-glutamine (HyClone) and 1.5-2.0 mM L-asparagine (Fluka Chemie AG, Buchs, Switzerland) and various concentrations of either GA or Erwinia L-asparaginase (Erwinase, ErAII) for 24 h. The kinetic parameters of the enzymes for the hydrolysis of L-asparagine were determined by a spectrophotometric method as described. 7,8 At pH 7.5, the K m of glycosylasparaginase, Erwinia L-asparaginase and Escherichia coli L-asparaginase was 538, 90 and 130 mM and V max 1.49, 16 and 23 mM/min, respectively. One unit of enzyme causes hydrolysis of 1 mmol of L-asparagine per minute under standard conditions.…”

A snapshot of the Ixodes scapularis degradome

“…The 3 putatively active enzymes in family T1, ISCW019233, ISCW006222 and ISCW05987 have been provisionally identified as proteosome catalytic subunits 1, 2 and 3, enzymes that are part of the protein turn over housekeeping mechanism in the cell (Gomes et al, 2006). In the case of the 3 enzymes in family T2, ISCW001950, ISCW000866 and ISCW001928, they have been provisionally identified as glycosylasparaginase precursor, threonine type isoaspartyl dipeptidase and taspase-1, enzymes that play important role(s) in the metabolism of β-aspartyl peptides, cell proliferation (Noronkoski et al, 1998; Cantor et al, 2009). Of the 8 enzymes in family T3, 5 sequences ISCW000530, ICSW003582, ISCW009859, ISCW010660 and ISCW012371 have been provisionally identified as gamma-glutamyltransferase homologs, an enzyme that is present in cell membranes and is involved in cross cell membrane trafficking of amino acids and peptides as well as glutathione metabolism (Langford et al, 2010; Leggatt and Iwama, 2009).…”