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 × 10⁹ 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.