Differences in the chemical reactivity of individual molecules of an enzyme

Xue, Qifeng; Yeung, Edward S.

doi:10.1038/373681a0

Cited by 338 publications

(326 citation statements)

References 19 publications

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“…S2). Thus, in the present study, we could not detect intermolecular (static) variation as in other single molecule enzyme studies (14)(15)(16). Instead, the variability observed here is due to the limited number of events we could record from individual molecules.…”

Section: Two Dwell Time Populations Neither Resulted From Adp Accumulcontrasting

confidence: 63%

“…variation seen for other enzymes (14)(15)(16). Kinetic modeling suggested that the two dwell time populations originate from two active site conformers with different ATP binding features.…”

Section: Significancementioning

confidence: 86%

“…Here we explored the feasibility to study ATPase kinetics from individual myosin molecules by total internal reflection fluorescence (TIRF) microscopy using fluorescently labeled ATP as used in previous studies to follow ATP turnover by motor proteins (10)(11)(12)(13). One aim was to see whether myosin molecules show a similar variability in kinetic parameters among individual molecules as previously found in single molecule studies for other enzymes (14)(15)(16). Such variability among individual molecules would limit, even in single molecule studies, detection of subtle but relevant changes in ATPase kinetics by, for example, posttranslational modifications.…”

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confidence: 99%

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ATP turnover by individual myosin molecules hints at two conformers of the myosin active site

Amrute-Nayak¹,

Lambeck²,

Radocaj³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Coupling of ATP hydrolysis to structural changes in the motor domain is fundamental to the driving of motile functions by myosins. Current understanding of this chemomechanical coupling is primarily based on ensemble average measurements in solution and muscle fibers. Although important, the averaging could potentially mask essential details of the chemomechanical coupling, particularly for mixed populations of molecules. Here, we demonstrate the potential of studying individual myosin molecules, one by one, for unique insights into established systems and to dissect mixed populations of molecules where separation can be particularly challenging. We measured ATP turnover by individual myosin molecules, monitoring appearance and disappearance of fluorescent spots upon binding/dissociation of a fluorescent nucleotide to/from the active site of myosin. Surprisingly, for all myosins tested, we found two populations of fluorescence lifetimes for individual myosin molecules, suggesting that termination of fluorescence occurred by two different paths, unexpected from standard kinetic schemes of myosin ATPase. In addition, molecules of the same myosin isoform showed substantial intermolecular variability in fluorescence lifetimes. From kinetic modeling of our two fluorescence lifetime populations and earlier solution data, we propose two conformers of the active site of myosin, one that allows the complete ATPase cycle and one that dissociates ATP uncleaved. Statistical analysis and Monte Carlo simulations showed that the intermolecular variability in our studies is essentially due to the stochastic behavior of enzyme kinetics and the limited number of ATP binding events detectable from an individual myosin molecule with little room for static variation among individual molecules, previously described for other enzymes.single ATP turnover assay | ATP dwell times | dwell time distribution | TIRF microscopy F orces and movements, generated when myosins interact with actin filaments, are driven by the coupling of conformational changes in the myosin head domain to particular steps of the ATP hydrolysis cycle (1-3). Essential steps of the ATP hydrolysis cycle for skeletal muscle myosin and acto-myosin were characterized in solution studies (1,4,5). With the concept of Hill (6), it became possible to relate solution studies to mechanical, biochemical, and structural studies on muscle fibers (7, 8) to form a general concept for the coupling of ATP hydrolysis to the generation of mechanical work (6, 9).In studies on large ensembles of myosin molecules such as solution and fiber studies, however, crucial reaction steps could be masked by the ensemble averaging and thus may complicate relating solution kinetics to structural changes and the generation of forces and movements. In addition, ensemble studies can be complicated by mixed populations of myosin molecules-for example, by the presence of different myosin isoforms or different posttranslational modifications. Here we explored the feasibility to study ATPase kinetics from ...

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Section: Two Dwell Time Populations Neither Resulted From Adp Accumulcontrasting

confidence: 63%

Section: Significancementioning

confidence: 86%

mentioning

confidence: 99%

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ATP turnover by individual myosin molecules hints at two conformers of the myosin active site

Amrute-Nayak¹,

Lambeck²,

Radocaj³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Further analysis using higher order correlation functions did not help discrimination between this simple model involving two alternative conformations and another involving a continuum of conformers. 195 This seminal study definitely confirmed the existence of static and dynamic heterogeneity in enzymes, 196,197 illustrating the power of single-molecule fluorescence studies for elucidating enzymatic reaction mechanisms inaccessible via ensemble methods.…”

Section: Quenching Due Tomentioning

confidence: 85%

Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

Michalet¹,

Weiss²,

Jaeger³

2006

ChemInform

125

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“…Ideally, each transition to another inhibition state will be reflected in a 25% change of substrate turnover. It is known, however, that the individual activities of different enzyme molecules can vary by up to a factor of four, even without inhibitor (1,3), and it is possible that the activity of an individual enzyme may fluctuate upon inhibitor binding and release. In this case, it would not be possible to distinguish the different states of inhibition depicted in Scheme 1 by comparing the substrate turnover…”

mentioning

confidence: 99%

Stochastic inhibitor release and binding from single-enzyme molecules

Gorris

Rissin

Walt

2007

Proc. Natl. Acad. Sci. U.S.A.

116

133

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Inhibition kinetics of single-␤-galactosidase molecules with the slow-binding inhibitor D-galactal have been characterized by segregating individual enzyme molecules in an array of 50,000 ultrasmall reaction containers and observing substrate turnover changes with fluorescence microscopy. Inhibited and active states of ␤-galactosidase could be clearly distinguished, and the large array size provided very good statistics. With a pre-steady-state experiment, we demonstrated the stochastic character of inhibitor release, which obeys first-order kinetics. Under steady-state conditions, the quantitative detection of substrate turnover changes over long time periods revealed repeated inhibitor binding and release events, which are accompanied by conformational changes of the enzyme's catalytic site. We proved that the rate constants of inhibitor release and binding derived from stochastic changes in the substrate turnover are consistent with bulk-reaction kinetics.␤-galactosidase ͉ enzyme kinetics ͉ fluorescence microscopy ͉ single molecule T he emergence of new assays for studying enzymes at the single-molecule level has profoundly extended our view of enzyme mechanisms. Whereas stochastic molecular behaviors are hidden by using bulk methods, single-molecule experiments have revealed that, in a population of enzymes, such as lactate dehydrogenase (1), phosphatase (2), or ␤-galactosidase (3), the catalytic rates of individual enzyme molecules are heterogeneous and do not interconvert quickly. Furthermore, it has been shown that the substrate turnover of individual ␤-galactosidase (4), cholesterol oxidase (5), or lipase (6) molecules undergoes dynamic fluctuations in sequential catalytic cycles. These two variations in enzyme activity are referred to as static and dynamic heterogeneity, respectively, and have both been attributed to different conformational states of the enzyme. Despite such heterogeneity, the Michaelis-Menten model derived from bulk enzyme experiments still holds for single-molecule experiments once a stochastic perspective is adopted (4,7,8).Traditionally, one of the most important tools to elucidate an enzyme's catalytic mechanism has been to study the enzyme in the presence of inhibitors. Here, we set out to correlate inhibition mechanisms established in bulk enzyme studies with singleenzyme molecule experiments. To date, inhibitors have been used in single-molecule studies to obtain information about conformational dynamics. Ha et al. (9) showed that it is possible to distinguish between free and inhibitor-bound states of a single-staphylococcal nuclease enzyme molecule. The binding of an inhibitor imposed a conformational constraint on the enzyme molecule resulting in a change of single-molecule polarization and intramolecular single-pair FRET. In this article, we report the direct observation of inhibitor release and binding from single-enzyme molecules by monitoring their substrate turnover.

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Differences in the chemical reactivity of individual molecules of an enzyme

Cited by 338 publications

References 19 publications

ATP turnover by individual myosin molecules hints at two conformers of the myosin active site

ATP turnover by individual myosin molecules hints at two conformers of the myosin active site

Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

Stochastic inhibitor release and binding from single-enzyme molecules

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