Single Enzyme Studies Reveal the Existence of Discrete Functional States for Monomeric Enzymes and How They Are “Selected” upon Allosteric Regulation

Nikos S. Hatzakis, Li Wei, Sune Klamer Jørgensen, Andreas Hjarne Kunding, Pierre-Yves Bolinger, Nicky Ehrlich, Ivan Makarov, Michael Skjot, Allan Svendsen, Per Hedegård, Dimitrios Stamou

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Abstract

Allosteric regulation of enzymatic activity forms the basis for controlling a plethora of vital cellular processes.
While the mechanism underlying regulation of multimeric enzymes is generally well understood and proposed to
primarily operate via conformational selection, the mechanism underlying allosteric regulation of monomeric enzymes is
poorly understood. Here we monitored for the first time allosteric regulation of enzymatic activity at the single molecule
level. We measured single stochastic catalytic turnovers of a monomeric metabolic enzyme (Thermomyces lanuginosus
Lipase) while titrating its proximity to a lipid membrane that acts as an allosteric effector. The single molecule measurements revealed the existence of discrete binary functional states that could not be identified in macroscopic measurements due to ensemble averaging. The discrete functional states correlate with the enzyme’s major conformational states and are redistributed in the presence of the regulatory effector. Thus, our data support
allosteric regulation of monomeric enzymes to operate via selection of preexisting functional states and not via induction of ones.
Original languageEnglish
JournalJournal of the American Chemical Society
Volume134
Issue number22
Pages (from-to)9296-9302
ISSN0002-7863
DOIs
Publication statusPublished - 2012

Bibliographical note

Copyright © 2012 American Chemical Society

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