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Redox cycling and kinetic analysis of single molecules of solution-phase nitrite reductase.

ABSTRACT: Single-molecule measurements are a valuable tool for revealing details of enzyme mechanisms by enabling observation of unsynchronized behavior. However, this approach often requires immobilizing the enzyme on a substrate, a process which may alter enzyme behavior. We apply a microfluidic trapping device to allow, for the first time, prolonged solution-phase measurement of single enzymes in solution. Individual redox events are observed for single molecules of a blue nitrite reductase and are used to extract the microscopic kinetic parameters of the proposed catalytic cycle. Changes in parameters as a function of substrate concentration are consistent with a random sequential substrate binding mechanism.

SUBMITTER: Goldsmith RH

PROVIDER: S-EPMC3198337 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Redox cycling and kinetic analysis of single molecules of solution-phase nitrite reductase.

Goldsmith Randall H RH Tabares Leandro C LC Kostrz Dorota D Dennison Christopher C Aartsma Thijs J TJ Canters G W GW Moerner W E WE

Proceedings of the National Academy of Sciences of the United States of America 20111003 42

Single-molecule measurements are a valuable tool for revealing details of enzyme mechanisms by enabling observation of unsynchronized behavior. However, this approach often requires immobilizing the enzyme on a substrate, a process which may alter enzyme behavior. We apply a microfluidic trapping device to allow, for the first time, prolonged solution-phase measurement of single enzymes in solution. Individual redox events are observed for single molecules of a blue nitrite reductase and are use ...[more]

PMID: 21969548

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