Carbohydrate-binding property of peptide: N-glycanase from mouse fibroblast L-929 cells as evaluated by inhibition and binding experiments using various oligosaccharides.
Carbohydrate binding to peptide: N-glycanase from mouse fibroblast L-929 cells (L-929 PNGase) and inhibition by oligosaccharides of its catalytic activity were studied. L-929 PNGase was found to bind strongly with oligosaccharides having triomannosido-N,N'-diacetyl-chitobiosyl (Man3GlcNAc2) structure (Kd = approximately 10 microM). This binding was inhibited by mannotriose (Man3; Man alpha 1-->3[Man alpha 1-->6]Man) but not by N,N'-diacetylchitobiose (GlcNAc2; GlcNAc beta 1-->4GlcNAc). Scatchard analysis indicated that there exist two binding sites for Man3 on a homodimeric form of a 105-kDa subunit. Oligosaccharides having Man3GlcNAc2 structure were also shown to be strong inhibitors for the PNGase-catalyzed reaction (Ki = approximately 10 microM). The minimum structural requirements for inhibition of the PNGase activity were Man3 and GlcNAc2. Enzyme kinetic studies showed that the mechanism of inhibition by the oligosaccharides and Man3 fits well with a model wherein two inhibitor binding sites reside on L-929 PNGase. The conformity of Kd with IC50 values may be taken as an evidence for inhibition of the catalytic activity by the oligosaccharides and Man3 through the occupation of the binding sites with these molecules. On the other hand, inhibition by GlcNAc2 followed the simple competitive mode. Since the minimum substrate for the L-929 PNGase was shown to be Man beta 1-->4GlcNAc beta 1-->4GlcNAc beta 1-->peptide, GlcNAc2 may be directly accessible to the catalytic site in competition with substrate. Interestingly, alkylation of -SH group in L-929 PNGase caused complete loss of the catalytic activity, but the carbohydrate binding activity was completely retained, indicating that the catalytic site(s) is discriminated from the carbohydrate-binding sites in the active site of this enzyme. The carbohydrate-binding property seems to be unique to soluble PNGases from mammals and may be associated not only with regulation of the enzyme activity, but also with receptor and carrier functions for glycoconjugates in certain intracellular processes.
Suzuki
T
T
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Japan.
Kitajima
K
K
Inoue
Y
Y
Inoue
S
S
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002240:Carbohydrate Sequence; D005347:Fibroblasts / Q000201:enzymology; D007700:Kinetics; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000494:pharmacology*; D009844:Oligosaccharides / Q000378:metabolism* / Q000494:pharmacology*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000378:metabolism; D013379:Substrate Specificity
D009005:Monosaccharides; D009844:Oligosaccharides; D011134:Polysaccharides; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase