Determination of enzyme mechanisms by radiationless energy transfer kinetics.
Rigorous definition of the elementary steps of an enzymatic reaction requires visualization of transient enzyme-substrate (ES) complexes. Measurement of radiationless energy transfer (RET) between enzyme tryptophan residues and a fluorescent dansyl (5-dimethylaminonaphthalene-1-sulfonyl) substrate provides a sensitive means to observe ES complexes directly. Analysis of the rate of formation and breakdown of ES complexes by RET can serve as the basis of a rapid kinetic approach to enzyme mechanisms. Both pre-steady-state and steady-state kinetics can be performed in the same RET experiment. Analysis at steady state precisely determines k(cat) and K(m) values by multiple means. Analysis at pre-steady state determines the number of intermediates, the type of reaction mechanism, and all the individual binding and rate constants. Chymotrypsin was chosen as a standard of reference for RET kinetics because extensive investigations have established both the existence of transient intermediates in the course of its catalytic process and the range of values to be expected for pertinent kinetic constants. As predicted, RET kinetics readily detects the two known intermediates in the alpha-chymotrypsincatalyzed hydrolysis of specific ester substrates. The results are both qualitatively and quantitatively in accord with data derived for this enzyme from classical kinetics. Hence, this experimental study both validates and demonstrates the theoretical advantages and potential of RET kinetics. The generality of the approach has been investigated by synthesizing a family of dansyl-labeled substrates designed to meet the specificity requirements of a number of metallo- and nonmetallo- exo- and endopeptidases. In all cases, the ES complex is observed readily at micromolar or lower concentrations of enzyme under stopped-flow conditions. The success of the RET kinetic approach on proteolytic enzymes shows its broad utility.
D002268:Carboxypeptidases / Q000378:metabolism; D002918:Chymotrypsin / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D007700:Kinetics; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
D002268:Carboxypeptidases; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin