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

Abstract

Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis and selfpowered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. Experimental observations of catalysisenhanced enzyme diffusion, to date, have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an Anti-Brownian ELectrokinetic (ABEL) trap and in-solution spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an Anti-Brownian ELectrokinetic (ABEL) trap and in-solution single-particle tracking (SPT), we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ~20% enhancement seen in parallel FCS experiments using p-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte-Carlo simulations, we establish that pNPP-induced dye blinking at the ~10 ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models--including those of our own--in the field, and indicate that in-solution SPT and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.

Master curve of boosted diffusion for 10 catalytic enzymes

“…One notable case is the enzyme alkaline phosphate, which catalyzes a highly exothermic reaction with H =−43.5 kJmol -1 yet positive G = +30.2 (28) or +61.5 (29) kJmol -1 at a high turnover rate. This shows no detected enhanced diffusion in our study, in agreement with a recent earlier study (21). Therefore, we consider it more meaningful physically to seek correlation with G, the maximum available work that can be extracted from the reaction (15,16).…”

“…The other enzymes -aldolase, phosphoglucoisomerase, pyruvate kinase, hexokinase, and phosphofructokinase, were selected from the glucose cascade cycle (25). Among the three endergonic reactions (G > 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.…”

“…Moreover, measuring boosted enzymatic motion has proved to be 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)(20)(21)(22) as has been reviewed critically (23).…”

Nanopore electro-osmotic trap for the label-free study of single proteins and their conformations

“…Scientists have developed new types of single-molecule traps aiming to overcome such shortcomings. For example, the anti-Brownian electro-kinetic (ABEL) trap 1,2 can hold a dye-labeled protein for seconds using real-time electro-kinetic feedback control. Electrostatic fluidic traps were shown to trap unfolded dye-labeled proteins under very low salt conditions, and to detect their charge 3 .…”

Perspective: a stirring role for metabolism in cells

“…It has been suggested that experimental artifacts affecting FCS may be responsible for the high diffusion coefficient values reported in the literature (Günther et al , 2018; Feng & Gilson, 2019). Along these lines, alternative techniques such as nuclear magnetic resonance (Günther et al , 2019), dynamic light scattering (Zhang et al , 2018), anti‐Brownian electrokinetic trapping (Chen et al , 2020), and single‐molecule displacement mapping (Choi et al , 2022) have found no diffusion enhancement for some of these enzymes, namely, aldolase and alkaline phosphatase (Riedel et al , 2015; Illien et al , 2017). Nevertheless, other techniques, such as single‐molecule measurements using total internal reflection fluorescence microscopy, have reported an even higher diffusion coefficient for urease than the value obtained with FCS (Xu et al , 2019).…”