SUBSTRATE-SPECIFICITY AND REACTION-MECHANISM OF HUMAN GLYCOASPARAGINASE - THE N-GLYCOSIDIC LINKAGE OF VARIOUS GLYCOASPARAGINES IS CLEAVED THROUGH A REACTION-MECHANISM SIMILAR TO L-ASPARAGINASE

Human glycoasparaginase (N4-(beta-N-acetyl-D-glucosaminyl)-L-asparagin ase, EC 3.5.1.26) hydrolyzes a series of compounds that contain L-aspa ragine residue with free alpha-amino and alpha-carboxyl groups. Substr ates include high mannose and complex type glycoasparagines as well as those that lack the di-N-acetylchitobiose moiety, L-aspartic acid bet a-methyl ester and L-aspartic acid beta-hydroxamate. The enzyme is ina ctive toward L-asparagine and L-glutamine and glycoasparagines contain ing substituted alpha-amino and/or alpha-carboxyl groups. In the prese nce of the acyl acceptor hydroxylamine, glycoasparaginase catalyzes th e synthesis of L-aspartic acid beta-hydroxamate from aspartyl-glucosam ine, L-aspartic acid beta-methyl ester, and L-aspartic acid. C-13 NMR studies using O-18-labeled L-aspartic acid demonstrate that glycoaspar aginase catalyzes an oxygen exchange between water and the carboxyl gr oup at C-4 of L-aspartic acid. These results indicate that glycoaspara ginase reacts as an exo-hydrolase toward the L-asparagine moiety of th e substrates and the free alpha-amino and alpha-carboxyl groups are re quired for the enzyme reaction. The results are consistent with an L-a sparaginase-like reaction pathway which involves a beta-aspartyl enzym e intermediate. Since glycoasparaginase is active toward a series of s tructurally different glycoasparagines, we suggest the revised systema tic name of N4-(beta-glycosyl)-L-asparaginase for the enzyme.