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

Abstract

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^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.