Irreversible inhibitors and activity-based probes as research tools in chemical glycobiology.

In this review, we will discuss the enzymes that are involved in the synthesis and degradation of glycoconjugates and we will give an overview of the inhibitors and activity-based probes (ABPs) that have been used to study these. Following discussion of some general aspects of the biosynthesis and degradation of N-linked glycoproteins, attention is focused on the enzymes that hydrolyze the protein-carbohydrate linkage, peptide N-glycanase and glycosylasparaginase and their mechanism. We then focus on the biosynthesis of O-linked glycoproteins and glycolipids and in particular on the enzymes that hydrolyze the interglycosidic linkages in these, the glycosidases. Some important mechanism-based glycosidase inhibitors that form a covalent bond with the targeted enzyme(s), their corresponding ABPs and their application to study this class of enzymes are highlighted. Finally, alternative pathways for degradation of glycoconjugates and an ABP-based strategy to study these will be discussed.

Glycosylasparaginase inhibition studies: competitive inhibitors, transition state mimics, noncompetitive inhibitors.

Glycosylasparaginase catalyzes the hydrolysis of the N-glycosylic bond between asparagine and N-acetylglucosamine in the catabolism of N-linked glycoproteins. Previously only three competitive inhibitors, one noncompetitive inhibitor, and one irreversible inhibitor of glycosylasparaginase activity had been reported. Using human glycosylasparaginase from human amniotic fluid, L-aspartic acid and four of its analogues, where the alpha-amino group was substituted with a chloro, bromo, methyl or hydrogen, were competitive inhibitors having Ki values between 0.6-7.7 mM. These results provide supporting evidence for a proposed intramolecular autoproteolytic activation reaction. A proposed phosphono transition state mimic and a sulfo transition state mimic were competitive inhibitors with Ki values 0.9 mM and 1.4 mM, respectively. These results support a mechanism for the enzyme-catalyzed reaction involving formation of a tetrahedral high-energy intermediate. Three analogues of the natural substrate were noncompetitive inhibitors with Ki values between 0.56-0.75 mM, indicating the presence of a second binding site that may recognize (substituted)acetamido groups.

The first demonstration of a procaryotic glycosylasparaginase.

Glycosylasparaginase was purified to near homogeneity from intracellular lysates of Flavobacterium meningosepticum. The enzyme is a heterodimer with an estimated molecular weight of 38 kDa and consists of one alpha-subunit (18 kDa) and one beta-subunit (16 kDa). The beta-subunit of the Flavobacterium enzyme has a direct evolutionary relationship to the beta-subunit of mammalian glycosylasparaginases as evidenced by: (1) strong cross-reactivity with antibodies made to the denatured rat beta-subunit, (2) a high degree of homology with the amino-terminus of the corresponding eukaryotic enzymes, and (3) irreversible inactivation with 5-diazo-4-oxo-L-norvaline, a reagent known to react with the catalytic amino-terminal threonine residue on the beta-subunit of a mammalian glycosylasparaginase.

Aspartylglycosaminuria: protein chemistry and molecular biology of the most common lysosomal storage disorder of glycoprotein degradation.

Aspartylglycosaminuria (AGU) (McKusick 20840) is the most common disorder of glycoprotein degradation caused by the failure of lysosomes to digest the protein-to-carbohydrate linkage of Asn-linked glycoproteins. During the past few years there has been significant progress in our understanding of both the protein chemistry and molecular biology of glycosylasparaginase (EC 3.5.1.26) as well as the molecular changes underlying the storage disease AGU that results from deficiency of this lysosomal hydrolase. Modern clinical assays have been developed for the diagnosis and carrier detection of this disease. Detailed structure, substrate specificity, mechanism of action, and a part of the active site of glycosylasparaginase have been defined. Molecular biology of glycosylasparaginase has progressed rapidly and already some mutations in the glycosylasparaginase gene resulting in AGU have been identified. Evolutionary aspects based on sequence data indicate a mechanistic relationship between mammalian glycosylasparaginases and bacterial/plant asparaginases.

Glycosaparaginase from human leukocytes. Inactivation and covalent modification with diazo-oxonorvaline.

The apparent active site of human leukocyte glycoasparaginase (N4-(beta-acetylglucosaminyl)-L-asparaginase EC 3.5.1.26) has been studied by labeling with an asparagine analogue, 5-diazo-4-oxo-L-norvaline. Glycoasparaginase was purified 4,600-fold from human leukocytes with an overall recovery of 12%. The purified enzyme has a Km of 110 microM, a Vmax of 34 mumol x l-1 x min-1, and a specific activity of 2.2 units/mg protein with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. The carbohydrate content of the enzyme is 15%, and it exhibits a broad pH maximum between 7 and 9. The 88-kDa native enzyme is composed of 19-kDa light (L) chains and 25-kDa heavy (H) chains and it has a heterotetrameric structure of L2H2-type. The glycoasparaginase activity decreases rapidly and irreversibly in the presence of 5-diazo-4-oxo-L-norvaline. At any one concentration of the compound, the inactivation of the enzyme is pseudo-first-order with time. The inhibitory constant, K1, is 80 microM and the second-order rate constant 1.25 x 10(3) M-1 min-1 at pH 7.5. The enzyme activity is competitively protected against this inactivation by its natural substrate, aspartylglucosamine, indicating that this inhibitor binds to the active site or very close to it. The covalent incorporation of [5-14C]diazo-4-oxo-L-norvaline paralleled the loss of the enzymatic activity and one inhibitor binding site was localized to each L-subunit of the heterotetrameric enzyme. Four peptides with the radioactive label were generated, purified by high performance liquid chromatography, and sequenced by Edman degradation. The sequences were overlapping and all contained the amino-terminal tripeptide of the L-chain. By mass spectrometry, the reacting group of 5-diazo-4-oxo-L-norvaline was characterized as 4-oxo-L-norvaline that was bound through an alpha-ketone ether linkage to the hydroxyl group of the amino-terminal amino acid threonine.

Effect of different compounds on 1-aspartamido-beta-N-acetylglucosamine amidohydrolase from human liver.

The effect of varous compounds on 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was studied. N-Acetylcysteine inhibited the nezyme non-competitively (Ki 3.2 mM), whereas 3-hydroxybutanone inhibited competitively (Ki 4.1 mM). Methionine, isoleucine and cystathionine apparently enhanced the enzyme activity. The enzyme had a mol. wt. of 63000 as determined by gel filtration. The present studies differentiate between the aspartylglucosylaminase from human liver and that obtained from various other sources.