Kinetics of Iterative Carbohydrate Transfer to Polysaccharide Catalyzed by Chondroitin Polymerase on a Highly Sensitive Flow-Type 27 MHz Quartz-Crystal Microbalance

• Published in: Chemistry : a European journal 2012-06, Vol.18 (24), p.7388-7393
• Main Authors: Mori, Toshiaki, Kodera, Takayuki, Yoshimine, Hiroshi, Kakuta, Yoshimitsu, Sugiura, Nobuo, Kimata, Koji, Okahata, Yoshio
• Format: Article
• Language: English
• Subjects: Carbohydrates; Catalysis; Chemistry; chondroitin polymerase; E coli; Elongation; enzymatic glycosylation; Glucuronic Acid - chemistry; Glucuronic Acid - metabolism; Glycosylation; Hexosyltransferases - metabolism; Kinetics; Microbalances; Models, Chemical; polymerization; Polysaccharides; Polysaccharides - chemistry; Polysaccharides - metabolism; Quartz; Quartz - metabolism; Quartz crystal microbalance; Quartz crystals; Strain analysis
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.201200342

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. One‐sugar transfer: Kinetics of one‐sugar transfer can be evaluated by using a highly sensitive flow‐type quartz‐crystal microbalance. Binding affinity and turnover number for UDP‐GlcA and UDP‐GalNAc are quite different. The catalytic efficiency is similar for GlcA transfer and GalNAc transfer (see scheme).