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

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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 (kon), translational movement (ktr), and dissociation (koff) with single-molecule fluorescence imaging. The kon for a single chitin microfibril was 2.1 × 109 M−1 μm−1 s−1. The koff showed two components, kfastoff (3.2 s−1, 78%) and kslowoff (0.38 s−1, 22%), corresponding to bindings to different crystal surfaces. From the kon, kfastoff, kslowoff 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.

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

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Publication details

The article was received on 09 Jul 2017, accepted on 24 Oct 2017 and first published on 24 Oct 2017


Article type: Paper
DOI: 10.1039/C7CP04606E
Citation: Phys. Chem. Chem. Phys., 2017, Advance Article
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    Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis

    A. Nakamura, T. Tasaki, Y. Okuni, C. Song, K. Murata, T. Kozai, M. Hara, H. Sugimoto, K. Suzuki, T. Watanabe, T. Uchihashi, H. Noji and R. Iino, Phys. Chem. Chem. Phys., 2017, Advance Article , DOI: 10.1039/C7CP04606E

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