Kinetics of Iterative Carbohydrate Transfer to Polysaccharide Catalyzed by Chondroitin Polymerase on a Highly Sensitive Flow‐Type 27 MHz Quartz‐Crystal Microbalance |
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| Authors: | Prof Dr Toshiaki Mori Takayuki Kodera Hiroshi Yoshimine Prof Dr Yoshimitsu Kakuta Prof Dr Nobuo Sugiura Prof Dr Koji Kimata Prof Dr Yoshio Okahata |
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| Institution: | 1. Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori‐ku, Yokohama 226‐8501 (Japan), Fax: (+81)?45‐924‐5836;2. Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology (PRESTO), 4‐1‐8 Honcho Kawaguchi, Saitama 332‐0012 (Japan);3. Department of Biochemistry, Kyushu University, Fukuoka 812‐8581 (Japan);4. Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute 480‐1195 (Japan) |
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| 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 (Kd=0.97 μM ). Although the binding affinity and the catalytic rate constant for each monomer were considerably different, the apparent catalytic efficiency (kcat/Km) was similar (6.3×104 M ?1 s?1 for GlcA transfer and 3.4×104 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. |
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| Keywords: | chondroitin polymerase enzymatic glycosylation kinetics polymerization quartz‐crystal microbalance |
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