Kinetics of alpha-mannosidase action on various alpha-D-mannopyranosyl linkages in hen ovalbumin glycopeptides as monitored by carbon 13 nuclear magnetic resonance spectroscopy.
We show that 13C NMR spectroscopy is a practical method for following the kinetics of enzymatic digestion of individual carbohydrate residues of glycopeptides and for determining the structures of the products of partial digestions. Specifically, we study the jack bean alpha-mannosidase digestion of the two hen ovalbumin glycopeptides Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6(Man alpha 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to Asn (Compound A) and Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to Asn (Compound B). The reported "rule" that jack bean alpha-mannosidase hydrolyzes Man alpha 1 leads to 2Man and Man alpha 1 leads to 6Man linkages at least 15 times faster than Man alpha 1 leads to 3Man linkages (Tai, T., Yamashita, K., Ogata-Arakawa, M., Koide, N., Muramatsu, T., Iwashita, S., Inoue, Y., and Kobata, A. (1975) J. Biol. Chem. 250, 8569-8575) is not of general validity. Although the Man alpha 1 leads to 2Man (alpha) linkage of Compound A is the first one to be digested, the Man alpha 1 leads to 3Man (beta) linkage is hydrolyzed next, faster than the Man alpha 1 leads to 6Man (alpha) linkage. The Man alpha 1 leads to 3Man (alpha) linkage is hydrolyzed very slowly. We show that it is practical to use 13C NMR spectroscopy to determine when the enzymatic digestion should be halted to isolate derivatives such as Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to Asn and Man alpha 1 leads to 3Man alpha 1 leads to 6Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to Asn.
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002645:Chickens; D005260:Female; D006020:Glycopeptides; D006027:Glycosides / Q000032:analysis*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008361:Mannosidases / Q000378:metabolism*; D008362:Mannosides / Q000032:analysis*; D010047:Ovalbumin; D043323:alpha-Mannosidase
D006020:Glycopeptides; D006027:Glycosides; D008362:Mannosides; D010047:Ovalbumin; D008361:Mannosidases; D043323:alpha-Mannosidase