1. Glycosylasparaginase activity requires the alpha-carboxyl group, but not the alpha-amino group, on N(4)-(2-Acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine
J M Risley, D H Huang, J J Kaylor, J J Malik, Y Q Xia, W M York Arch Biochem Biophys. 2001 Jul 15;391(2):165-70. doi: 10.1006/abbi.2001.2416.
Glycosylasparaginase catalyzes the hydrolysis of the N-glycosylic bond in N(4)-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine in the catabolism of N-linked oligosaccharides. A deficiency, or absence, of enzyme activity gives rise to aspartylglycosaminuria, the most common disorder of glycoprotein metabolism. The enzyme catalyzes the hydrolysis of a variety of asparagine and aspartyl compounds containing a free alpha-carboxyl group and a free alpha-amino group; computational studies suggest that the alpha-amino group actively participates in the catalytic mechanism. In order to study the importance of the alpha-carboxyl group and the alpha-amino group on the natural substrate to the reaction catalyzed by the enzyme, 14 analogues of the natural substrate were studied where the structure of the aspartyl group of the substrate was changed. The incremental binding energy (DeltaDeltaGb) for those analogues that were substrates was calculated. The results show that the alpha-amino group may be substituted with a group of comparable size, for the alpha-amino group contributes little, if any, to the transition state binding energy of the natural substrate. The alpha-amino group position acts as an "anchor" in the binding site for the substrate. On the other hand, the alpha-carboxyl group is necessary for enzyme activity; removal of the alpha-carboxyl group or changing it to an alpha-carboxamide group results in no hydrolysis reaction. Also, N-acetyl-D-glucosamine is not sufficient for binding to the active site for efficient hydrolysis by the enzyme. These results provide supporting evidence for a proposed intramolecular autoproteolytic activation reaction for the enzyme. However, the results raise a question as to an important role for the alpha-amino group in the catalytic mechanism as indicated in computational studies.
2. The molecular and crystal structures of 4-N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine trihydrate and 4-N-(beta-D-glucopyranosyl)-L-asparagine monohydrate. The x-ray analysis of a carbohydrate-peptide linkage
L T Delbaere Biochem J. 1974 Oct;143(1):197-205. doi: 10.1042/bj1430197.
X-ray analyses have shown that the glucopyranose rings of GlcNAc-Asn [4-N-(2-acetamido-2-deoxy-beta-d-glucopyranosyl)-l-asparagine] and Glc-Asn [4-N-(beta-d-glucopyranosyl)-l-asparagine] both have the C-1 chair conformation and also that the glucose-asparagine linkage of each molecule is present in the beta-anomeric configuration. The dimensions (the estimated standard deviations of the last digit are in parentheses) of the glycosidic bond in GlcNAc-Asn and Glc-Asn are, respectively, C((1))-N((1)) 0.1441(6)nm, 0.146(2)nm; angle O((5))-C((1))-N((1)) 106.8(3) degrees , 105.7(8) degrees ; angle C((2))-C((1))-N((1)) 111.1(4) degrees , 110.4(9) degrees ; angle C((1))-N((1))-C((9)) 121.4(4) degrees , 120.5(9) degrees . The glycosidic torsion angle C((9))-N((1))-C((1))-C((2)) is 141.0 degrees and 157.6 degrees in GlcNAc-Asn and Glc-Asn respectively. Hydrogen-bonding is extensive in these two crystal structures and does affect one torsion angle in particular. Two very different values of chi(1)(N-C(alpha)-C(beta)-C(gamma)) occur for the asparagine residue of the two different molecules; the values of chi(1), -69.0 degrees in GlcNAc-Asn and 61.9 degrees in Glc-Asn, correspond to two different staggered conformations about the C(alpha)-C(beta) bond as the NH(3) (+) group is adjusted to different hydrogen-bonding patterns. The two trans-peptide groups in GlcNAc-Asn show small distortions in planarity whereas that in Glc-Asn is more non-planar. The mean plane through the atoms of the amide group at C((2)) in GlcNAc-Asn is approximately perpendicular (69 degrees ) to the mean plane through the C((2)), C((3)), C((5)) and O((5)) atoms of the glucose ring and that at C((1)) is less perpendicular (65 degrees ). The mean plane through the atoms of the amide group in Glc-Asn makes an angle of only 55 degrees with the mean plane through these same four atoms of the glucose ring. The N((1))-H bond of the amide at C((1)) is trans to the C((1))-H bond in these two compounds; the N((2))-H bond of the amide at C((2)) is trans to the C((2))-H bond in GlcNAc-Asn. The values of the observed and final calculated structure amplitudes have been deposited as Supplementary Publication SUP 50035 (26 pages) at the British Library (Lending Division), (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.
3. Synthesis of N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine analogues: succinamide, L-2-hydroxysuccinamide, and L-2-hydroxysuccinamic acid hydrazide analogues
H De Huang, J M Risley Carbohydr Res. 2000 Nov 17;329(3):487-93. doi: 10.1016/s0008-6215(00)00224-x.
The syntheses of three analogues of N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine are described. N-(2-Acetamido-2-deoxy-beta-D-glucopyranosyl)succinamide was synthesized by the reaction of pentafluorophenyl succinamate with 2-acetamido-2-deoxy-beta-D-glucopyranosylamine. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosylamine was synthesized, and the complete assignment of the 1H NMR spectrum is given. Reaction of the protected beta-D-glycosylamine with L-malic acid chloralid in the presence of a coupling agent (EEDQ) gave N4-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-L-malamic acid chloralid that was deprotected two ways: (1) using ammonia, which gave N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-2-hydroxysuccinamide, and (2) using hydrazine, which gave N4-(2-acetamido-2-deoxy-1-D-glucopyranosyl)-L-2-hydroxysuccinamic acid hydrazide.
