Rapid kinetic analysis of mechanochemical adenosinetriphosphatases.

The analysis of a reaction pathway by stopped-flow and chemical quench-flow methods provides the only means of directly measuring the rates of reaction at the active site of an enzyme. The interpretation of data from each technique is similar and involves solution of the differential equations describing the approach of the intermediates to steady state. In the case of the stopped-flow data, I have tried to show how intuition can simplify some of the math to allow the solution of more complex models and to reveal the form of the concentration dependence of the rates. In the case of the chemical quench-flow data, more effort is expended in collecting one time course and accordingly more time is spent in fitting the data directly to a given model. One often is restricted by the quantities of enzyme available so that it is feasible to perform chemical quench-flow experiments at only one or two carefully selected concentrations of substrate. Stopped-flow and chemical quench-flow experiments complement one another because each provides information that is inaccessible, or at least difficult to obtain, by the other method. Our current chemical quench-flow apparatus, requiring only 20 microliter of sample per time point, should allow many more enzymes to be examined by this method.