2018年1月31日
Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis.
Physical chemistry chemical physics : PCCP
- 巻
- 20
- 号
- 5
- 開始ページ
- 3010
- 終了ページ
- 3018
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1039/c7cp04606e
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, 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.
- ID情報
-
- DOI : 10.1039/c7cp04606e
- ISSN : 1463-9076
- PubMed ID : 29090301