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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Alcaligenes / enzymology
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Alcaligenes / genetics
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Amino Acid Substitution
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Bacterial Proteins / chemistry
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Copper / chemistry
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Fluorescence Resonance Energy Transfer
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Kinetics
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Microfluidic Analytical Techniques
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Models, Molecular
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Mutagenesis, Site-Directed
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Nitrite Reductases / chemistry*
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Nitrite Reductases / genetics
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Nitrite Reductases / metabolism*
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Oxidation-Reduction
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Protein Structure, Quaternary
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Solutions
Substances
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Bacterial Proteins
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Recombinant Proteins
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Solutions
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Copper
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Nitrite Reductases