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Citation: XU Yan, SUN Le-le, GAO Yan-Jing, QIN Wei-Wei, PENG Tian-Huan, LI Di. Research Progresses in Single Molecule Enzymology[J]. Chinese Journal of Analytical Chemistry, ;2016, 44(9): 1437-1446. doi: 10.11895/j.issn.0253-3820.160201 shu

Research Progresses in Single Molecule Enzymology

Division of Physical Biological & Bioimaging Center, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China

Received Date: 18 March 2016
Revised Date: 18 April 2016

Fund Project: This work was supported by the National Natural Science Foundation of China (Nos. 21227804, 21373260, 31371015), the Youth Innovation Promotion Association, Chinese Academy of Sciences (No.2013174), and the Open Project of State Key Laboratory of Oral Diseases (No.SKLOD2015OF05).

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The single molecule imaging and technologies that developed in 1990s have successfully probed the dynamics of single molecule enzyme catalysis in real time in vitro. Ever since then, single molecule enzymology has entered the golden age of rapid developing. Individual features of each enzyme hidden in the overall average have been discovered, and many new catalytic mechanisms have been proposed. Single molecule enzymology sheds light on the dynamic interactions between enzymes and substrates or products, deepening the understanding of biochemical reactions. This review described the recent research progresses of single molecule protease and ribozyme.
- Single molecule,
- Enzyme,
- Ribozyme,
- Fluorescence resonance energy transfer,
- Fluorescence microscopy,
- Review
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Figure 1. (A) Scheme of cholesterol oxidase (COx) catalysis. When COx catalyzing its substrate to generate product,the flavin adenine dinucleotide (FAD) containing in active site is reduced to the reduced flavin adenine dinucleotide,FADH2,then re-oxidized by O₂,corresponding changes of its fluorescent state. (B) Single molecule COx fluorescence emission intensity changes with time. (C) On-time distribution of single COx with 2 mmol/L substrate. (D) The 2D conditional probability distribution of On-time separated by certain numbers of turnovers. (E) The autocorrelation analysis of On-time. (F) Proposed simple model of enzyme catalysis.Fig. 1 was reproduced with permission from Ref ^[9],Copyright 1998 The American Association for the Advancement of Science.

Figure 2. (A) Scheme for study of horseradish peroxidase (HRP) activity fluctuation： a,immobilization scheme of HRP；b,correlation analysis of fluorescence intensity changes of single HRP catalysis； (B) Product release process study of HRP： a,scheme for immobilization of HRP; b,Correlation plots of the lifetime,anisotropy,and intensity on single HRP-catalyzed Amplex Red fluorogenic assay; c,Theory model of two kinds of product releasing processes; (C) Conformational manipulation of HRP through single molecule magnetic microscopy: a,the conceptual scheme of the experimental system; b,the catalytic rates changes under external force; c,conceptual scheme of conformational fluctuation of single enzyme protein when being deformed by external force. Fig. 2(A) was reproduced with permission from Ref ^[12],copyright 1999 Elsevier Limited. Fig. 2(B) was reproduced with permission from Ref ^[13],copyright 2014 American Chemical Society. Fig. 2(C) was reproduced with permission from Ref ^[14],copyright 2015 National Academy of Sciences.

Figure 3. (A) A virus-based study of HRP catalysis: a,experiment scheme for HRP capsuled in virus capsid,substrates diffuse into the cavity of the virus capsid,and are catalyzed by HRP; b,autocorrelation analysis of HRP catalysis. (B) Study of HRP catalysis revealing a two-step radical dismutation reaction mechanism: a,scheme for glass optical fiber bundle confined single HRP catalysis study; b,averaged catalysis turnover rate for single HRP detection; c,averaged catalysis turnover rate in bulk measurement. (C) Allosteric inhibition study of product generated by HRP: a,scheme for HRP trapped by liposome; b,a typical time trace of fluorescence intensity from HRP catalyzing product formation inside single liposome vesicle as a function of time; c,simple model for noncompetition inhibition and the correlation function between initial reaction rate of individual HRP and the number of product molecules needed for completely inhibition.Fig. 3(A) was reproduced with permission from Ref ^[15],copyright 2007 Nature Publishing Group. Fig. 3(B) was reproduced with permission from Ref ^[16],copyright 2010 American Chemical Society. Fig. 3(C) was reproduced with permission from Ref ^[17],copyright 2012 National Academy of Sciences.

Figure 4. (A) Ras activation by Son of Sevenless: a,ras loaded with fluorescent labeled GTP is trapped on to supported bilayer,after catalyzed by SOS,the resulted fluorescent GDP is released to solution. (B) Specific interaction of anaphase-promoting complex (APC) and substrate: a,fluorescent labeled substrate is fixed onto the interface,meanwhile ubiquitin and APC are also fluorescently labeled,and the interaction of APC and substrate are monitored by fluorescence microscopy; b,processive affinity amplification enhances the specificity of APC. (C) Substrate degradation by the proteasome: a,construction of substrate with different ubiquitin configurations; b,single molecule study strategy,the proteasome is trapped on the coverslips,the substrate is conjugated with different ubiquitin conformers,and when the substrate interacting with proteasome,fluorescent spot could be recorded.(A) was reproduced with permission from Ref ^[22],copyright 2014The American Association for the Advancement of Science. (B) was reproduced with permission from Ref ^[23],copyright 2015 The American Association for the Advancement of Science. (C) was reproduced with permission from Ref ^[24],copyright 2015The American Association for the Advancement of Science.

Figure 5. (A) Single-molecule study of ribozyme catalysis and folding:a,structural model of ribozyme and experimental scheme;b,the model of P1 interaction with the active center of ribozyme; c,time histogram of folding into native structure. (B) Study of structural dynamics and function: a,single-molecule and bulk solution measurements of enzymatic activities; b,the undocking kinetics is complex,suggesting four docked states of distinct stabilities. c,Docked states have a strong memory effect. (C) Study of modification showing impacts on catalysis: a,exemplary single-molecule FRET time trajectories of the major subpopulations of the WT and variant hairpin ribozyme-noncleavable substrate analog complexes;b,experimental and simulated cleavage time courses of WT and variant ribozymes. (D) Molecular crowding Accelerates Ribozyme Docking and Catalysis: a,PEG stabilizes the docked conformation of the hairpin ribozyme; b,PEG helps the ribozyme fold in near physiological conditions; c,PEG accelerates ribozyme cleavage.(A) was reproduced with permission from Ref ^[25],copyright 2000,The American Association for the Advancement of Science. (B) was reproduced with permission from Ref ^[28],copyright 2002,The American Association for the Advancement of Science. (C) was reproduced with permission from Ref ^[29],copyrights 2004,National Academy of Sciences. (D) was reproduced with permission from Ref ^[30],copyright 2014 American Chemical Society.