Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy

A method for sensitively monitoring enzyme kinetics and activities by using dual color fluorescence crosscorrelation spectroscopy is described, This universal method enables the development of highly sensitive and precise assays for real-time kinetic analyses of any catalyzed cleavage or addition reaction, where a chemical linkage is formed or cleaved through an enzyme's action between two fluorophores that can be discriminated spectrally, In this work a homogeneous assay with restriction endonuclease EcoRI and a 66-bp double-stranded DNA containing the GAATTC recognition site and fluorophores at each 5' end is described. The enzyme activity can be quantified down to the low picomolar range (>1.6 pM) where the rate constants are linearly dependent on the enzyme concentrations over two orders of magnitude. Furthermore, the reactions were monitored online at various initial substrate concentrations in the nanomolar range, and the reaction rates were clearly represented by the Michaelis-Menten equation with a K-M of 14 +/- 1 nM and a k(cat) of 4.6 +/- 0.2 min(-1). In addition to kinetic studies and activity determinations, it is proposed that enzyme assays based on the dual-color fluorescence cross-correlation spectroscopy will be very useful for high-throughput screening and evolutionary biotechnology.