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), translational movement (k), and dissociation (k) with single-molecule fluorescence imaging. The k for a single chitin microfibril was 2.1 × 10 M μm s. The k showed two components, k (3.2 s, 78%) and k (0.38 s, 22%), corresponding to bindings to different crystal surfaces. From the k, k, k and ratio of fast and slow dissociations, dissociation constants for low and high affinity sites were estimated as 2.0 × 10 M μm and 8.1 × 10 M μm, respectively. The k was 52.5 nm s, and processivity was estimated as 60.4. The apparent inconsistency between high turnover (52.5 s) calculated from k and biochemically determined low k (2.6 s) is explained by a low ratio (4.8%) of productive enzymes on the chitin surface (52.5 s × 0.048 = 2.5 s). Our results highlight the importance of single-molecule analysis in understanding the mechanism of enzymes acting on a solid-liquid interface.