Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis.

Serratia marcescens chitinase A is a linear molecular motor that hydrolyses crystalline chitin in a processive manner. Here, we quantitatively determined the rate constants of elementary reaction steps, including binding (k _on ), translational movement (k _tr ), and dissociation (k _off ) with single-molecule fluorescence imaging. The k _on for a single chitin microfibril was 2.1 × 10 ⁹ M ^-1 μm ^-1 s ^-1 . The k _off showed two components, k (3.2 s ^-1 , 78%) and k (0.38 s ^-1 , 22%), corresponding to bindings to different crystal surfaces. From the k _on , k, k and ratio of fast and slow dissociations, dissociation constants for low and high affinity sites were estimated as 2.0 × 10 ^-9 M μm and 8.1 × 10 ^-10 M μm, respectively. The k _tr was 52.5 nm s ^-1 , and processivity was estimated as 60.4. The apparent inconsistency between high turnover (52.5 s ^-1 ) calculated from k _tr and biochemically determined low k _cat (2.6 s ^-1 ) is explained by a low ratio (4.8%) of productive enzymes on the chitin surface (52.5 s ^-1 × 0.048 = 2.5 s ^-1 ). Our results highlight the importance of single-molecule analysis in understanding the mechanism of enzymes acting on a solid-liquid interface.