title: Kinetics of iterative carbohydrate transfer to polysaccharide catalyzed by chondroitin polymerase on a highly sensitive flow-type 27 MHz quartz-crystal microbalance.
abstract: Using a highly sensitive flow-type 27 MHz quartz crystal microbalance, we could detect a small mass change during stepwise and alternating one-sugar transfer of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) to an acceptor, catalyzed by chondroitin polymerase from Escherichia coli strain K4 (K4CP), and analyze the elongation mechanism of K4CP. K4CP was found to bind strongly to a chondroitin acceptor (K(d)=0.97 μM). Although the binding affinity and the catalytic rate constant for each monomer were considerably different, the apparent catalytic efficiency (k(cat)/K(m)) was similar (6.3×10(4)  M(-1)  s(-1) for GlcA transfer and 3.4×10(4)  M(-1)  s(-1) for the GalNAc transfer). This is reasonable for the smooth alternating elongation of GlcA and GalNAc on the acceptor. This is the first study to report the determination of kinetic parameters for enzymatic, alternated, sugar elongation.
authors.affiliation: Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. tmori@bio.titech.ac.jp
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Mori
authors.forename: Takayuki
authors.initials: T
authors.lastname: Kodera
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yoshimine
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Kakuta
authors.forename: Nobuo
authors.initials: N
authors.lastname: Sugiura
authors.forename: Koji
authors.initials: K
authors.lastname: Kimata
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Okahata
mesh_terms: D002384:Catalysis; D020723:Glucuronic Acid / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006602:Hexosyltransferases / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011791:Quartz / Q000378:metabolism
chemical_list: D011134:Polysaccharides; D011791:Quartz; D020723:Glucuronic Acid; D006602:Hexosyltransferases; C460368:chondroitin polymerase, E coli