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Mass Spectrometry: Isotope Effect

Mass Spectrometry: Isotope Effect

Most elements exist in nature as a mixture of isotopes. The isotopes differ in weight due to their respective number of neutrons. The molecular weight of a molecule is different depending on the specific isotope of its elements involved. As a result, the mass spectrum of the molecule exhibits peaks from the same fragment at multiple positions. The positions of these mass signals depend on the mass differences between isotopes. Furthermore, the intensity of these signals is dependent on the...

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Related Experiment Video

Updated: May 14, 2026

Isotopic Effect in Double Proton Transfer Process of Porphycene Investigated by Enhanced QM/MM Method

Isotopic Effect in Double Proton Transfer Process of Porphycene Investigated by Enhanced QM/MM Method

Published on: July 19, 2019

Enzymatic single-molecule kinetic isotope effects.

Christopher R Pudney¹, Richard S K Lane, Alistair J Fielding

¹Manchester Institute of Biotechnology and Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.

Journal of the American Chemical Society

|February 14, 2013

Summary

Single molecule kinetic isotope effects (KIEs) using spFRET reveal enzyme reaction details. This method separates enzymatic H-transfer from protein and fluorophore dynamics, advancing catalysis understanding.

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Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

Published on: January 16, 2016

Area of Science:

Biochemistry
Enzyme kinetics
Physical chemistry

Background:

Ensemble measurements of kinetic isotope effects (KIEs) have advanced understanding of enzyme-catalyzed reactions but face limitations.
KIEs are crucial for studying rate-limiting steps, quantum tunneling, dynamics, and multiple reactive states in enzymes.

Purpose of the Study:

To explore single molecule (SM) enzymatic KIEs for novel insights into enzyme catalysis.
To apply single pair fluorescence energy transfer (spFRET) for measuring SM KIEs in H-transfer reactions.

Main Methods:

Utilized single pair fluorescence energy transfer (spFRET) to measure SM KIEs.
Developed and evaluated methods for extracting SM KIEs from spFRET time traces.
Investigated H-transfer catalyzed by pentaerythritol tetranitrate reductase.

Main Results:

Successfully measured SM enzymatic KIEs for H-transfer reactions.
Demonstrated the ability of SM KIE analysis to differentiate between enzymatic and nonenzymatic processes.
Separated contributions from protein dynamics, fluorophore behavior, and the H-transfer reaction itself.

Conclusions:

Single molecule KIE analysis provides a powerful new approach to study enzyme catalysis.
This method allows for the deconvolution of reaction chemistry from intrinsic protein dynamics.
Offers a pathway to resolve long-standing controversies in enzyme kinetics.