Single-Molecule Enzymology: Critical Aspects Exemplified by Myosin ATPase Activity

The myosin ATPase provides a model system for developing single-molecule enzyme assays because of its slow turnover rate and low Km for fluorescent ATP analogs. While the average lifetime of fluorescence spots arising from Cy3-nucleotide bound to single immobilised myosin sites is close to that expected from the catalytic activity measured in bulk preparations, quantitative comparisons suggest other processes may contribute to fluctuations. Here we review the possible factors involved. We also discuss the benefits and difficulties of measuring the same molecule over repeated cycles. The potential complexity of these assays is illustrated by an example in which Cy3-labelled substrates are apparently turned over by two different myosin molecules in the same bulk environment, but they show inherently different kinetics.