Single-Molecular Enzymatic Elongation of Hyaluronan Polymers Visualized by High-Speed Atomic Force Microscopy

• Published in: Journal of the American Chemical Society 2012-12, Vol.134 (50), p.20254-20257
• Main Authors: Mori, Toshiaki, Hirose, Atsushi, Hagiwara, Tatsuya, Ohtsuka, Masanori, Kakuta, Yoshimitsu, Kimata, Koji, Okahata, Yoshio
• Format: Article
• Language: English
• Subjects: Catalysis; Glucuronosyltransferase - chemistry; Hyaluronan Synthases; Hyaluronic Acid - chemistry; Lipid Bilayers; Microscopy, Atomic Force - methods; Pasteurella multocida - enzymology; Polymers - chemistry
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja309646s

Using high-speed scanning atomic force microscopy, we directly observed single-molecular enzymatic elongation of hyaluronan polymer chains at intervals of 10 s on a mica or lipid bilayer surface, on which Pasteurella multocida hyaluronic acid synthase (pmHAS) was immobilized. The reaction was started by the addition of both UDP-glucuronic acid and UDP-N-acetylglucosamine monomers. The average catalytic elongation rate constant (k cat) was found to be 1.8 mer s–1 from one active enzyme physically adsorbed on a mica surface. When pmHAS was immobilized by inserting its hydrophobic tail part into lipid bilayers, most of the enzymes retained their activity, and the k cat values were found to be in the range 1–10 mer s–1 for 29 enzymes (average was k cat = 2–4 mer s–1). These k cat values were lowest level of k cat = 1–100 s–1 obtained in bulk solution by radioisotope methods.