The effect of the protein matrix on glycan processing in glycoproteins. Kinetic analysis of three rat liver Golgi enzymes.
In order to assess the basis for the regulatory effects of the protein matrix on the processing of glycans in glycoproteins, we have used the avidin-biotinylglycan neoglycoprotein model system to compare the kinetic parameters for three rat liver Golgi enzymes acting on their free and protein-bound glycan substrates. Two modes of glycan display in the avidin complex were produced by the use of either the biotinyl- or the 6-biotinamidohexanoyl-group as ligands for the avidin binding. N-Acetylglucosaminyltransferase I gave a 100-fold decrease in Vmax/Km for the avidin complex of Man5GlcNAc2-(biotinyl)Asn as compared to the free glycan derivative; the rate difference reflects a large (25x) decrease in the Vmax and a relatively small increase (4x) in Km. When the substrate with the extension arm (Man5GlcNAc2-(6-biotinamidohexanoyl)Asn) was used, the difference between Vmax/Km for free and avidin-bound substrate was only 6-fold. The Vmax/Km ratio for N-acetylglucosaminyltransferase II also showed a 10-fold difference for free and avidin-bound GlcNAcMan3GlcNAc2-(biotinyl)Asn; the introduction of the extension arm in the complex reduced the difference to about 3-fold. The third enzyme, galactosyltransferase, acting on the substrate GlcNAcMan5-GlcNAc2-R in the presence of the mannosidase II-inhibitor swainsonine, showed a small, 2- to 3-fold, decrease in the Vmax for the bound substrates, both with and without the extension arm. The results suggest that the protein matrix affects the catalytic efficiency rather than the substrate affinity of the processing enzymes.
Shao
M C
MC
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
Wold
F
F
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001360:Avidin / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism; D005964:Glucosyltransferases / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D017351:N-Acetylglucosaminyltransferases; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D013379:Substrate Specificity; D017026:Swainsonine
D000470:Alkaloids; D006023:Glycoproteins; D011134:Polysaccharides; D001360:Avidin; D001710:Biotin; D005700:Galactosyltransferases; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase; D017026:Swainsonine