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

摘要

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-acetylglucos-amine 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.