Facile variation of reagent concentrations in rapid quench enzymology

Abstract

Detailed enzymological investigations are essential in elucidating the reaction pathway of an enzyme, including the identification of reaction intermediates, the quantitative determination of the rate-determining step(s), and the nature of the transition state. The information derived from these enzymological studies is especially important in the development of specific inhibitors that can find application in both research applications and as potential drugs. Many enzymological studies are performed using steady-state kinetics, as, for example, in determining relative affinities in high-throughput screens, determining the order of substrate binding and product release, and structure-activity relationships (SAR) [1]. However, steady-state kinetics are limited in that they only provide the kcat and Km parameters that are potentially complex functions of all the individual rate constants in the enzymatic reaction pathway. Therefore, transient state kinetics have been developed to elucidate both presteady-state and one-turnover reactions allowing for a more complete description of the individual reaction steps [2]. When possible, enzymologists attempt to develop enzyme assays that use an absorbance or fluorescent signal allowing for continuous measurement of product formation (or substrate depletion). The number of enzyme reactions that can be monitored directly using native substrates is extremely limited and has led to the use of artificial substrates that incorporate elements with detectable extinction coefficients or fluorescent properties or coupled assays. However, artificial substrates and coupling enzymes do not adequately address the needs of enzymology. First, full characterization of an enzyme requires work with native substrates and most native substrates do not have useful absorbance or fluorescent properties. Second, coupled assays are not useful in transient state kinetics where the lag time in observation of product formation through the coupling enzymes would mask the reaction of interest. Because of these limitations, enzymologists have for many years relied on rapid quench flow. In this technique, individual time points are collected along the reaction course and then analyzed by a suitable technique, often using radioactive labeling and separation of products from substrates. In order to perform these experiments rapidly enough to measure reaction kinetics on the millisecond time scale, a number of rapid quench apparatus have been developed. A rapid quench flow experiment generally involves three syringes. A number of manufacturers have specialized in making high-quality instruments capable of performing rapid quench flow on a microvolume basis, sufficiently small in volume to be useful for the enzymologist. These companies include Hi-Tech Scientific, Bio-Logic, and KinTek. One of the most common experiments performed by an enzymologist is the variation of substrate and/or enzyme concentration to determine dependencies of reaction rates on these two key variables. In many experiments, a wide-ranging substrate and/or enzyme concentration-dependence determination is not needed. For example, in a so-called single-turnover experiment, one may only be interested in determining whether one is working under conditions of limiting association between enzyme and substrate or limiting rate of reaction on the enzyme (i.e., actual chemistry) [2]. This can easily be determined by doubling the enzyme and Analytical Biochemistry 312 (2003) 80–83