Detailed studies on substrate structure requirements of glycoamidases A and F.
Glycoamidases (peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, EC 3.5.1.52; also known as peptide: N-glycanases (PNGases) release N-linked oligosaccharides from glycopeptides and/or glycoproteins by hydrolyzing the glycosylated beta-amide bond of the asparagine side chain. The most widely used glycoamidases are those from Flavobacterium meningosepticum (glycoamidase F or PNGase F) and almond emulsin (glycoamidase A or PNGase A). To study the substrate structure requirement of these enzymes systematically, we synthesized >30 glycopeptides containing cellobiose, lactose, GlcNAc, and di-N,N'-acetylchitobiose (CTB). The length of the peptide was varied from one to five amino acids, and glycosylamines were linked to either Asn or Gln located at different positions in the peptide, including NH2 and COOH termini. Neither enzyme could cleave cellobiose and lactose glycopeptides, indicating that the 2-acetamido group on the Asn-linked GlcNAc is important in the recognition by both glycoamidases A and F. GlcNAc peptides could be cleaved by both enzymes, albeit not as effectively as CTB glycopeptides. Neither enzyme requires the Asn-Xaa-(Ser/Thr) sequence (required for N-glycosylation) for activity. Glycoamidase A could even hydrolyze a Gln-bound CTB glycopeptide, whereas the action of glycoamidase F on this substrate is minimal. While glycoamidase A could act on CTB dipeptides, glycoamidase F preferred a tripeptide or longer. The Km and Vmax values of glycoamidase A for t-butoxycarbonyl-(CTB)-Asn-Ala-Ser-OMe were 2.1 mM and 0.66 micromol/min/mg, respectively. A natural glycodipeptide, Man9-GlcNAc2-Asn-Phe, was also completely hydrolyzed by glycoamidase A.
Fan
J Q
JQ
Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Lee
Y C
YC
D000581:Amidohydrolases / Q000378:metabolism*; D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D005417:Flavobacterium / Q000201:enzymology*; D006020:Glycopeptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D015394:Molecular Structure; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D006020:Glycopeptides; D007527:Isoenzymes; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase