Stopped-flow radiationless energy transfer kinetics: direct observation of enzyme-substrate complexes at steady state

Measurement of radiationless energy transfer (RET) between enzyme tryptophan residues and a fluorescent dansyl (5-dimethylaminonaphthalene-1-sulfonyl) substrate under stopped-flow conditions forms the basis of a rapid and sensitive kinetic approach to delineation of enzyme mechanisms. Both the pre steady state and steady state can be studied on one experiment. The ES complexes of even rapidly turned over dansyl substrates are observed directly at enzyme concentrations of 10(-8)-10(-6) M. If [ET] < Km, a steady state of ES complex formation and breakdown can be achieved and maintained even though the reaction is completed in a few seconds. RET kinetic analysis under stopped-flow steady-state conditions both simplifies and supplements conventional initial rate kinetic studies as illustrated here with bovine and yeast carboxypeptidases and alpha-chymotrypsin acting on Dns-(Gly)3-L-OPhe and Dns-(Gly)2-L-PheOMe, respectively. Since both the concentration and rate of breakdown of intermediates are observed, the kinetic parameters Kcat and Km can be determined precisely by multiple means. The capability of observing both steady-state and pre-steady-state RET kinetics in the same experiment greatly reduces errors in quantitative analysis, allowing a more rigorous definition of enzyme mechanisms.