By varying the time between mixing the reactants and adding the quenching reagent,
the kinetics of this build-up can be studied. A disadvantage of this approach is that it
uses more reactants than the stopped-flow method since the kinetic data are acquired
from a series of studies rather than by following one reaction for a period of time. Both
methods have difficulty in monitoring the first millisecond of reaction due to the need
to allow mixing to take place, but this problem can be partly solved by changing the
pH or temperature in order to slow down the reaction. Both methods commonly use
synthetic substrates that release a coloured product or give rise to a coloured acyl or
phosphoryl intermediate.Relaxation methods
The limitation of the stopped-flow method is the dead time during which the enzyme
and substrate are mixed. In the relaxation methods an equilibrium mixture of the
reactants is preformed and the position of equilibrium altered by a change in reaction
conditions. The most common procedure for achieving this is thetemperature jump
techniquein which the reaction temperature is raised rapidly by 5–10C by the
discharge of a capacitor or infrared laser. The rate at which the reaction mixture
adjusts to its new equilibrium (relaxation timet, generally a few microseconds) is
inversely related to the rate constants involved in the reaction. This return to equili-
brium is monitored by one or more suitable spectrophotometric methods. The
recorded data enable the number of intermediates to be deduced and the various rate
constants calculated from the relaxation times.
These pre-steady-state techniques have shown that the enzyme and its substrate(s)
associate very rapidly, with second-order rate constants for the formation of ES in the
range 10^6 to 10^8 M^1 s^1 and first-order rate constants for the dissociation of ES in
the range 10 to 10^4 s^1. The upper limit of these values is such that for some enzymes
virtually every interaction between an enzyme and its substrate leads to the formation
of a complex. The stopped-flow and quenching methods have also been used to study
other biochemical processes that are kinetically fast and may involve transient
intermediates. For example, the stopped-flow method has been applied to the study
of protein folding, protein conformational changes and receptor–ligand binding, and
the quenching method to the study of second messenger pathways (Section 17.4.1).Isothermal titration calorimetry
This is a general method for studying the thermodynamics of any binding (association)
process. It detects and quantifies small heat changes associated with the binding and has
the advantages of speed, accuracy and not requiring either of the reacting species to beCaption for fig. 15.12(cont.)
enter the delay line (variable volume between 25 and 1000 mm^3 ) and then the flow cell cuvette
with a minimum dead time of 0.6 ms. The flow can be stopped at any predetermined time
either by stopping the stepping motor or by closing the outlet from the reaction cuvette. The
reaction can be studied by visible, ultraviolet, fluorescence or circular dichroism spectroscopy.
The optical path length can be varied between 0.8 and 10 mm. In quench-flow mode the
minimum ageing time is<2 ms. The quenching agent is added from the third or fourth syringe.
(Reproduced by permission of BioLogic Science Instruments, France: website http://www.bio-logic.info.)607 15.3 Analytical methods for the study of enzyme reactions