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 (kon), translational movement (ktr), and dissociation (koff) with single-molecule fluorescence imaging. The kon for a single chitin microfibril was 2.1 × 10(9) M(-1) μm(-1) s(-1). The koff 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 kon, 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 ktr 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 ktr and biochemically determined low kcat (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.