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

Physical Chemistry Chemical Physics : PCCP
Akihiko NakamuraRyota Iino

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 konfor a single chitin microfibril was 2.1 × 109M-1μm-1s-1. The koffshowed 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 × 10-9M μm and 8.1 × 10-10M μm, respectively. The ktrwas 52.5 nm s-1, and processivity was estimated as 60.4. The apparent inconsistency between high turnover (52.5 s-1) calculated from ktrand 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.

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Citations

Jan 14, 2020·Protein Science : a Publication of the Protein Society·Benjamin A BaradJames S Fraser
Nov 12, 2020·Biophysics and Physicobiology·Akihiko NakamuraRyota Iino
Mar 21, 2019·Biochimica Et Biophysica Acta. General Subjects·Takayuki UmakoshiToshio Ando
Oct 14, 2021·Science Advances·Syeda Rubaiya NasrinAkira Kakugo

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Methods Mentioned

BETA
X-ray
atomic force microscopy
fluorescence imaging
affinity
PCR
fluorescence microscopy

Software Mentioned

IMOD
3dmod viewer

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