Spin‐exchange NMR spectroscopy in studies of the kinetics of enzymes and membrane transport

Abstract

Spin-exchange NMR techniques enable the measurement of the rates of exchange of solutes between chemically or physically distinct sites in reactions taking place at chemical equilibrium. The time scale of the events that are able to be investigated lies in the neighbourhood of 1 s. The earliest studies in this area of NMR spectroscopy involved chemical reactions in vitro but the procedures have been adapted to the study of enzyme-catalysed reactions both in vitro and in vivo, and more recently to transmembrane exchange processes. The emphasis in this review is on the various types of spin-exchange experiments, the analysis of data derived from them, estimates of uncertainty in measured rate constants, and their shortcomings. Those methods given special attention are saturation transfer, two-dimensional exchange spectroscopy (2D EXSY), the 'accordion' experiment and 'overdetermined' one-dimensional exchange spectroscopy.

Correcting reaction rates measured by saturation-transfer magnetic resonance spectroscopy

“…Even so, both scan-time and SNR are at a premium in such studies, and the accuracy of the calculation of k would certainly improve if more measurements and/or data with better SNR could be practically acquired. Indeed, there are alternative methods of measuring reaction rates such as 1D or 2D exchange spectroscopy (EXSY) [10,[22][23][24] that do not use chemical-selective irradiation and hence do not suffer from spillover effects. However, combining 1D spatially localized in vivo 31 P MRS with 1D or 2D EXSY, leads to impractical patient scan times, especially where fully-relaxed acquisitions are required [10], these being avoided by FAST.…”

“…Indeed, there are alternative methods of measuring reaction rates such as 1D or 2D exchange spectroscopy (EXSY) [10,[22][23][24] that do not use chemical-selective irradiation and hence do not suffer from spillover effects. However, combining 1D spatially localized in vivo 31 P MRS with 1D or 2D EXSY, leads to impractical patient scan times, especially where fully-relaxed acquisitions are required [10], these being avoided by FAST. For saturation-transfer methods, the correction approaches described herein can be extended to proton spectra, other field strengths, and other protocols, requiring only recalculation of the polynomial fitting coefficients for the new range of fitting parameters and available measurements.…”

“…Since TR is usually fixed as a user-defined parameter for the FAST protocol, we will neglect its change. Subtracting a term Δk that depends on the relevant parameters, we obtain a corrected value of k, k corr : (9) Using polynomial basis functions, the correction is (10) where P k and P ω are second-order polynomials in their arguments. A second-order polynomial was found to give acceptable error levels while only minor improvements were achieved with higher orders.…”

“…Errors in the reaction rate and relaxation times arising from off-resonance and incomplete saturation effects have been reported [6,[10][11][12]. Strategies for correcting these errors have been suggested, which typically require additional acquisitions with the saturating irradiation completely turned-off [8,9,[13][14][15].…”

Ethanol transport in Zymomonas mobilis measured by using in vivo nuclear magnetic resonance spin transfer

“…The enormous potential of in vivo NMR spectroscopy holds not only for applications in the study of enzyme reactions but also for almost any membrane transport process (17,21,22), whether fast (time scale of about 0.01 to 10 s) or slow (time scale of about 1 min or more). Examples that used both time scales are given in this report.…”