Single-molecular enzymatic elongation of hyaluronan polymers visualized by high-speed atomic force microscopy

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.