Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments

Chen, Zhijie; Shaw, Alan; Wilson, H. A.; Woringer, Maxime; Darzacq, Xavier; Marqusee, Susan; Wang, Quan; Bustamante, Carlos

doi:10.1073/pnas.2006900117

Cited by 39 publications

(57 citation statements)

References 57 publications

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“…The other enzymes—aldolase, phosphoglucoisomerase, pyruvate kinase, hexokinase, and phosphofructokinase—were selected from the glucose cascade cycle ( 25 ). Among the three endergonic reactions ( ΔG >0), alkaline phosphatase and triosephosphate isomerase were selected, to repeat the measurements of an earlier study ( 14 , 21 ), and phosphoglycerate kinase was selected from the glucose cascade cycle. The substrate concentrations were selected to give reaction half-lives of a few minutes.…”

Section: Resultsmentioning

confidence: 99%

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Master curve of boosted diffusion for 10 catalytic enzymes

Jee

Tlusty

Granick

2020

Proc. Natl. Acad. Sci. U.S.A.

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Molecular agitation more rapid than thermal Brownian motion is reported for cellular environments, motor proteins, synthetic molecular motors, enzymes, and common chemical reactions, yet that chemical activity coupled to molecular motion contrasts with generations of accumulated knowledge about diffusion at equilibrium. To test the limits of this idea, a critical testbed is the mobility of catalytically active enzymes. Sentiment is divided about the reality of enhanced enzyme diffusion, with evidence for and against. Here a master curve shows that the enzyme diffusion coefficient increases in proportion to the energy release rate—the product of Michaelis-Menten reaction rate and Gibbs free energy change (ΔG)—with a highly satisfactory correlation coefficient of 0.97. For 10 catalytic enzymes (urease, acetylcholinesterase, seven enzymes from the glucose cascade cycle, and one other), our measurements span from a roughly 40% enhanced diffusion coefficient at a high turnover rate and negativeΔGto no enhancement at a slow turnover rate and positiveΔG. Moreover, two independent measures of mobility show consistency, provided that one avoids undesirable fluorescence photophysics. The master curve presented here quantifies the limits of both ideas, that enzymes display enhanced diffusion and that they do not within instrumental resolution, and has possible implications for understanding enzyme mobility in cellular environments. The striking linear dependence of ΔGfor the exergonic enzymes (ΔG<0), together with the vanishing effect for endergonic enzyme (ΔG>0), are consistent with a physical picture in which the mechanism boosting the diffusion is an active one, utilizing the available work from the chemical reaction.

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Section: Resultsmentioning

confidence: 99%

“…Moreover, measuring boosted enzymatic motion has proven challenging, as it is a rapid process occurring over length scales of a few nanometers. Some studies assert that the apparent enhancement originates from experimental artifacts ( 19 – 22 ), as has been reviewed critically ( 23 ).…”

mentioning

confidence: 99%

Master curve of boosted diffusion for 10 catalytic enzymes

Jee

Tlusty

Granick

2020

Proc. Natl. Acad. Sci. U.S.A.

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“…3 B), and derive the diffusion coefficient D jump-length from the kinetic model fitting following the procedures as described in Ref. [17] and Methods (circle, Fig. 3 C ).…”

Section: Comparison Of Msd Analysis and Jump-length Analysismentioning

confidence: 99%

“…In the first Spot-On analysis paper, the authors only tested D within the range of 0.5 ∼ 14.5 µm 2 /s [19], implying that this method might not be able to capture faster-diffusing particles. Despite that, follow-up papers have used the Spot-On analysis to measure diffusion rates as fast as ∼50 µm 2 /s, giving us confidence to try this method on our cases [17]. In summary, we examine the diffusion of active urease with and without urea using SLB/glycerol chamber at two viscosity regimes.…”

Section: B Diffusion Of Active Urease At Low Viscosity Regimementioning

confidence: 99%

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Direct Single Molecule Imaging of Enhanced Enzyme Diffusion

Ross

Valdez

et al. 2019

Phys. Rev. Lett.

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Recent experimental results have shown that active enzymes can diffuse faster when they are in the presence of their substrates. Fluorescence correlation spectroscopy (FCS), which relies on analyzing the fluctuations in fluorescence intensity signal to measure the diffusion coefficient of particles, has typically been employed in most of the prior studies. However, flaws in the FCS method, due to its high sensitivity to the environment, have recently been evaluated, calling the prior diffusion results into question. It behooves us to adopt complementary and direct methods to measure the mobility of enzymes in solution. Herein, we use a novel technique of direct single-molecule imaging to observe the diffusion of single enzymes. This technique is less sensitive to intensity fluctuations and gives the diffusion coefficient directly based on the trajectory of the enzymes. Our measurements recapitulate that enzyme diffusion is enhanced in the presence of its substrate and find that the relative increase in diffusion of a single enzyme is even higher than those previously reported using FCS. We also use this complimentary method to test if the total enzyme concentration affects the relative increase in diffusion and if enzyme oligomerization state changes during catalytic turnover. We find that the diffusion increase is independent of the total background concentration of enzyme and the catalysis of substrate does not change the oligomerization state of enzymes.

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Comparison of Measurements for Enhanced Diffusion Problem in Chemical Reaction Systems

Jee,

Wen,

Wang

2024

Chin. J. Chem.

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Comprehensive SummaryThe problem of molecular diffusion in the soup of chemical reactions attracts mounting interest across fields ranging from chemistry to biophysics to material science. Chemical reactions involve bond breakup and formation, whose time scale is typically on the orders of fs to ps, while molecular diffusion occurs at time scales of μs to ms. The two processes are often considered orthogonal, given the vastly different scales. The serial results show that the enzyme's diffusion is enhanced in a substrate‐dependent manner, which was further extended to small molecule reaction systems, challenging this classical paradigm. However, the results from different groups using different techniques do not quantitatively agree, and a general mechanism is yet to be understood. We summarize experimental studies on diffusion problems and seek to reconcile the interpretation with understanding the limits of measurement tools and the chemical nature of reaction systems. Understanding molecular diffusion in chemical reactions will provide fresh thoughts in designing chemical systems such as molecular machines that harvest work at the nanoscale in a controllable manner.

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Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments

Cited by 39 publications

References 57 publications

Master curve of boosted diffusion for 10 catalytic enzymes

Master curve of boosted diffusion for 10 catalytic enzymes

Direct Single Molecule Imaging of Enhanced Enzyme Diffusion

Comparison of Measurements for Enhanced Diffusion Problem in Chemical Reaction Systems

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