Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.1.1 (asparaginase)
 2,695 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Aspartylglucosaminidase (AGA) is a lysosomal asparaginase that participates in the breakdown of glycoproteins by cleaving the amide bond between the asparagine and the oligosaccharide chain. Active AGA is an (alphabeta)2 heterotetramer of two non-identical subunits that are cleaved proteolytically from an enzymatically inactive precursor polypeptide. On the basis of the three-dimensional structure recently determined by us, we have here mutagenized the putative active site amino acids of AGA and studied by transient expression the effect of targeted substitutions on the enzyme activity and catalytic properties of AGA. These analyses support the novel type of catalytic mechanism, suggested previously by us, in which AGA utilizes as the nucleophile the N-terminal residue of the beta subunit and most importantly its alpha-amino group as a base that increases the nucleophilicity of the OH group. We also provide evidence for autocatalytic activation of the inactive AGA precursor and putative involvement of active site amino acids in the proteolytic processing. The data obtained on the structure and function of AGA would indicate that AGA is a member of a recently described novel class of hydrolytic enzymes (amidohydrolases) sharing a common structural determinant in their three-dimensional structure and whose catalytic mechanisms with an N-terminal nucleophile seem basically to be similar.
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PMID:Functional analyses of active site residues of human lysosomal aspartylglucosaminidase: implications for catalytic mechanism and autocatalytic activation. 867 Jul 96

Aspartylglucosaminidase (AGA) is a lysosomal asparaginase that takes part in the ordered degradation of glycoproteins and a deficiency of which results in a lysosomal accumulation disease aspartylglucosaminuria in human. The mature enzyme consists of 24-kDa and 17-kDa subunits, which are both heterogeneously glycosylated. Activation of the enzyme from a single precursor polypeptide into two subunits is accomplished in the endoplasmic reticulum (ER). The relative lack of this proteolytic capacity in several tested high-producing expression systems has complicated the production of active recombinant enzyme in high quantities, which would be an alternative for purification of this molecule for crystallization. Consequently, the AGA enzyme has to be purified directly from cellular or tissue sources for crystallographic analysis. Here we describe a large-scale purification method to produce milligram amounts of homogeneous AGA from human leukocytes. The purified AGA enzyme represents a heterogeneous pool of molecules not only due to glycosylation, but also heterogeneity at the polypeptide level, as demonstrated here. We were able to isolate a homogeneous peptide pool that was successfully crystallized and preliminary X-ray data collected from the crystals. The crystals diffract well to 2.0 angstroms and are thus suitable for determination of the crystal structure of AGA.
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PMID:Large-scale purification and preliminary x-ray diffraction studies of human aspartylglucosaminidase. 898 1