Modulation of Kinetic Pathways of Enzyme–Substrate Interaction in a Microfluidic Channel: Nanoscopic Water Dynamics as a Switch

Abstract

Enzyme‐mediated catalysis is attributed to enzyme–substrate interactions, with models such as “induced fit” and “conformational selection” emphasizing the role of protein conformational transitions. The dynamic nature of the protein structure, thus, plays a crucial role in molecular recognition and substrate binding. As large‐scale protein motions are coupled to water motions, hydration dynamics play a key role in protein dynamics, and hence, in enzyme catalysis. Here, microfluidic techniques and time‐dependent fluorescence Stokes shift (TDFSS) measurements are employed to elucidate the role of nanoscopic water dynamics in the interaction of an enzyme, α‐Chymotrypsin (CHT), with a substrate, Ala‐Ala‐Phe‐7‐amido‐4‐methylcoumarin (AMC) in the cationic reverse micelles of benzylhexadecyldimethylammonium chloride (BHDC/benzene) and anionic reverse micelles of sodium bis(2‐ethylhexyl)sulfosuccinate (AOT/benzene). The kinetic pathways unraveled from the microfluidic setup are consistent with the “conformational selection” fit for the interaction of CHT with AMC in the cationic reverse micelles, whereas an “induced fit” mechanism is indicated for the anionic reverse micelles. In the cationic reverse micelles of BHDC, faster hydration dynamics (≈550 ps) aid the pathway of “conformational selection”, whereas in the anionic reverse micelles of AOT, the significantly slower dynamics of hydration (≈1600 ps) facilitate an “induced fit” mechanism for the formation of the final enzyme–substrate complex. The role of water dynamics in dictating the mechanism of enzyme–substrate interaction becomes further manifest in the neutral reverse micelles of Brij‐30 and Triton X‐100. In the former, the faster water dynamics aid the “conformational selection” pathway, whereas the significantly slower dynamics of water molecules in the latter are conducive to the “induced fit” mechanism in the enzyme–substrate interaction. Thus, nanoscopic water dynamics act as a switch in modulating the pathway of recognition of an enzyme (CHT) by the substrate (AMC) in reverse micelles.

Decoding the Kinetic Pathways toward a Lipid/DNA Complex of Alkyl Alcohol Cationic Lipids Formed in a Microfluidic Channel

“…The details of the TCSPC set up are described elsewhere. 19,20 The observed fluorescence transients are fitted using a nonlinear least-squares fitting procedure (software F900 from Edinburgh Instrument, U.K.) to a function composed of the convolution of the instrument response function with a sum of exponentials and quality of the fitting is judged by a reasonably good chi-square (χ 2 ∼ 0.95− 1.07) value. Fluorescence anisotropy was determined by the equation:…”

“…G is considered as a correction factor of the instruments, and the value of G is determined by using the long tail matching technique of the fluorescence transients. 19,20 The time-dependent fluorescence Stokes shift was determined from time-resolved emission spectrum (TRES). Details procedure of the construction of TRES was described elsewhere.…”

“…Assuming a steady state approximation for AB in Scheme 2, concentration of the product lipoplexes (C) can be described by the following equation. 19,20…”

“…The instrument response function (IRF) is observed ∼75 ps by using a 375 nm laser excitation source. The details of the TCSPC set up are described elsewhere. , The observed fluorescence transients are fitted using a nonlinear least-squares fitting procedure (software F900 from Edinburgh Instrument, U.K.) to a function composed of the convolution of the instrument response function with a sum of exponentials and quality of the fitting is judged by a reasonably good chi-square (χ 2 ∼ 0.95–1.07) value. Fluorescence anisotropy was determined by the equation: where I VV (t) and I VH (t) signify the parallel and perpendicular polarized gated fluorescence transients, respectively, recorded using a vertically polarized excitation light source.…”

“…For this purpose, we have employed a microfluidics (MF) platform − based on fluorescence microscopy and picosecond-resolved photophysical study to probe lipid/DNA complexation on a millisecond (ms) time scale. By adjusting the flow rates of the reactants (lipids and DNA) within the microchannel of a specially designed microfluidic (MF) chip, a sub-1-ms time resolution has been achieved for exploring the kinetic pathways in lipoplex formation.…”