Enzymatic Synthesis of the N-Glycosidic Bond by -Aspartylation of Glycosylamines 1 Ilkka Mononen,* ,2 Georgi I. Ivanov,² Ivanka B. Stoineva,² Tiina Noronkoski,* and Dimiter D. Petkov² *Department of Clinical Chemistry, Kuopio University Hospital, FIN-70211 Kuopio, Finland; and ²Laboratory of BioCatalysis, Institute of Organic Chemistry, Bulgarian Academy of Sciences, BG-1113 Sofia, Bulgaria Received November 27, 1995 Glycosylasparaginase (EC 3.5.1.26) is an amidase, which cleaves the N-glycosidic linkage during glycopro- tein degradation leading to the liberation of L-aspartic acid from various glycoasparagines. In this work we demonstrate that glycosylasparaginase is also capable of catalyzing the synthesis of the N-glycosidic bond by N--aspartylation of -glycosylamine using 1-amino-N-acetylglucosamine as the nucleophile and L-aspartic acid -methyl ester as the -aspartyl donor. Kinetic studies indicated that -glycosylamine has 1390-fold higher reactivity than water in the de--aspartylation of the -aspartylenzyme, indicative of the presence of a -gly- cosylamine binding sub-site at the substrate binding site of glycosylasparaginase. The reaction can be applied to glycosylasparaginase-catalyzed biosynthesis of novel glycoasparagines. © 1996 Academic Press, Inc. The covalent coupling of the carbohydrate and protein fragment of N-linked glycoprotein can be achieved by N-glycosylation a of a protein asparagine carboxamide or by N--aspartylation b of a -glycosylamine (Scheme 1). The formation of the N-glycosidic carbohydrate-protein linkage in eukaryotic cells occurs co- translationally by oligosaccharyltransferase-catalyzed transfer of a common oligosaccharide pre- cursor to L-asparagine residues at the N-glycosylation sites in the sequon -Asn-X-(Ser/Thr)- of the growing peptide chain (1,2). In theory, enzyme-catalyzed N--aspartylation of glycosylamines would provide an alternative route for the synthesis of N-linked glycopeptides (3–5), though this has never been reported in practice. Glycosylasparaginase (GA, 3 aspartylglucosaminidase, N 4 -(-N-acetyl-D-glucosaminyl)-L- asparaginase, EC 3.5.1.26) is a lysosomal enzyme, which cleaves the N-glycosidic linkage during the degradation of glycoproteins. A genetic deficiency in its activity leads to aspartylglycosamin- uria (AGU; McKusick 20840)—the most common disorder of glycoprotein degradation in humans, which is characterized by accumulation of aspartylglucosamine and other glycoasparagines in body fluids and tissues (6). Substrates of glycosylasparaginase include high mannose and complex type glycoasparagines as well as L-aspartic acid -methyl ester and -hydroxamate (7). Based on partitioning experiments and oxygen exchange between water and the -carboxyl group of L- aspartic acid, it was considered that the glycosylasparaginase mechanism of action involves the formation of a -aspartylenzyme, H-Asp(GA)-OH (7). Peptide bonds can be formed in suitable conditions in a kinetically controlled enzyme aminolysis of the corresponding acyl amino or amino acid esters (8). We report here of a kinetically controlled, glycosylasparaginase-catalyzed synthesis of the N-glycosidic bond in the formation of aspartyl- 1 This work was supported by grants from the Sigrid Juselius Foundation and Academy of Finland (I.M.). 2 Corresponding author: Department of Clinical Chemistry, Kuopio University Hospital, P.O. Box 1777, FIN-70211 Kuopio, Finland. Fax: +358-71-173-179. 3 Abbreviations: AGU, aspartylglycosaminuria; GA, glycosylasparaginase; H-Asn(GlcNAc)-OH, 2-acetamido-1-L-- aspartamido-1,2-dideoxy--D-glucose, aspartylglucosamine; H-Asp(GA)-OH, -aspartyl glycosylasparaginase; H- Asp(Me)-OH, L-aspartic acid -methylester; GlcNAc-NH 2 , 1-amino-N-acetylglucosamine. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 218, 510–513 (1996) Article No. 0091 510 0006-291X/96 $12.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.