chemically modified or immobilised. The apparatus consists of a pair of matched cells
(sample and reference) of approximately 2 cm^3 volume contained in a microcalorimeter
(Fig. 15.13a). One of the reactants (say the enzyme preparation) is added to the sample
cell and the ligand (substrate, inhibitor or effector) added via a stepper-motor-driven
syringe. The mixture is stirred to ensure homogeneity. The reference cell contains an
equal volume of reference liquid. A constant power of less than 1 mW is applied to the
reference cell. This directs a feedback circuit activating a heater attached to the sample
cell. The addition of the ligand solution causes a heat change due to the binding process
and the dilution of both the enzyme and ligand preparations. If the reaction is exother-
mic less energy is required to maintain the cell at constant temperature. If the reaction is
endothermic more energy is required. The power required to maintain a constant
temperature is recorded as a series of spikes as a function of time (Fig. 15.13b). Each
spike is integrated to givemcal s^1 and summed to give the total heat exchange per
injection. The study is repeated with a series of increasing ligand concentrations
and control experiments carried out replacing the ligand with buffer solution to allow
the heat exchange (H) associated solely with the addition of ligand to be calculated.
A plot is then made of enthalpy change against the molar ratio of the ligand to enzyme.
The plot is hyperbolic from which it is possible to calculate enthalpy, free energy, and
entropy changes associated with the ligand binding and hence the dissociation constant,
Kd, and stoichiometry of bindingn. Isothermal titration calorimetry has been success-
fully used in the study of the thermodynamics of the interconversion of protein
conformations and the elucidation of the mechanism of allosterism.15.3.4 Analytical methods forin vivostudies
The increasing importance of genome sequencing studies, particularly in the context of
drug development, has stimulated the development of techniques for the study of
enzymes in intact cells and whole organisms.In vitromethods have the disadvantage
that they lead to the disruption of organelles and micro-departments, commonly result
in the release of activators or inhibitors and invariably use assay conditions that are not
representative of thein vivosituation. One of the most successful analytical techniques
for studying enzymology in individual cells and in whole organisms is nuclear magnetic
resonance spectroscopy (NMR). This non-invasive technique allows the measurement of
steady-state metabolite concentrations and of metabolic flux using simple proton NMR,
or the redistribution of a^13 C label among glycolytic intermediates or the use of^31 PNMR
to measure ATP turnover and flux. Evidence for enzyme–enzyme interaction has been
obtained by studying conformational changes in the enzyme protein. This approach
requires the protein to be labelled in some appropriate way. One of the most attractiveCaption for fig. 15.13(cont.)
of the ligand whilst the syringe rotates to provide continuous mixing. Heat is added or removed from the
sample cell, as appropriate, and the associated power required to maintain constant temperature recorded
in units ofmcal s^1. (b) Data for the binding of 2^0 CMP to RNase. Top panel, energy exchange; bottom panel,
the binding isotherm from which the value ofn(1),Kd(0.85mM) andH(16.7 kcal mol^1 ) can be calculated.
(Reproduced by permission of MicroCal Europe, Milton Keynes, UK: website http://www.microcal.com.)609 15.3 Analytical methods for the study of enzyme reactions