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 fast off (3.2 s −1 , 78%) and k slow off (0.38 s −1 , 22%), corresponding to bindings to different crystal surfaces. From the k on , k fast off , k slow off 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. Single-molecule analysis revealed elementary reaction steps and low productive binding ratio of chitinase A.