2] Rapid quench kinetic analysis of polymerases, adenosinetriphosphatases, and enzyme intermediates

Abstract

Transient-state kinetic methods allow definition of the sequence of reactions, occurring at the active site of an enzyme, following substrate binding and leading to product release. It is precisely these reaction steps that define the elementary steps, accounting for the high fidelity of DNA polymerization or establishing the pathway, for coupling of adenosine triphosphate (ATP) hydrolysis to energy transduction by molecular motors. In each case, the analysis of the reactions, occurring at the active site of the enzyme, by single turnover kinetic methods, has allowed the definition of the reaction sequence. On the other hand, steady-state kinetic analysis only establishes the order of substrate binding and the order of product release; the steady-state kinetic parameters kcat and kcat/Km only define the maximum rate of substrate to product conversion and a lower-limit estimate of the rate of substrate binding, respectively. Steady-state kinetic analysis cannot address questions, regarding the pathway of events, occurring at the active site following substrate binding and prior to product release. This chapter discusses the methods of kinetic analysis used to define the sequence and rates of reactions catalyzed by DNA polymerases and by force-transducing adenosinetriphosphatases (ATPases) and gives a brief explanation of the detection of enzyme intermediates. The methods are of general utility, in studying enzyme reaction mechanisms, and have led to the discovery of several new enzyme intermediates.