Many tools have been used to study the TIM mechanism, including X-ray crystallography and NMR, site-directed mutagenesis, and affinity labeling. Strong evidence for the mechanism, however, was supplied by studies using isotopic labeling of substrates. It was found that if the above reaction was carried out in tritiated water, one atom of tritium was stereospecifically incorporated into DHAP.
This result suggested that a base abstracts a proton from the substrate, and the proton then undergoes exchange with labeled protons from the solvent before being added back to form the
One unresolved questionwas whether only a single protein base was involved (so that the transfer was from substrate to base and directly back to form product) or whether a different base was responsible for protonation as part of a more extensive proton relay. The nature of the protein base was explored by doing a similar experiment to the one described above but in the other direction; that is, by labeling the DHAP and observing its conversion to G3P. Although the equilibrium lies far to the side of the DHAP, trapping by irreversible oxidation by G3P dehydrogenase of any G3P formed was used to convert significant quantities of DHAP. If the DHAP was labeled at C1, a small but measurable amount of the label was transferred to C2.
This result suggested that a single base was involved in the proton abstraction/proton addition step. If more than one base were involved, the chance that any label would not be washed out by the solvent and would be added to the deprotonated intermediate would be vanishingly small. In combination with other kinds of experiments, isotopic labeling was therefore invaluable in elucidating the mechanism of triosephosphate isomerase (shown below) in which B is a protein-derived base.
Isotopes can be used in another way to measure the energy barrier heights for various steps in the catalytic mechanism as noted above for the reaction catalyzed by dihydrofolate reductase. For example, if a proton transfer is involved in the rate-limiting step, then substitution of that proton with one of the heavier isotopes of hydrogen (deuterium or tritium) will cause the step to proceed more slowly. These so-called kinetic isotope effect experiments in combination with steady-state rate measurements in the case of TIM allowed the elucidation of the rate constants for partitioning of the cis-enediol intermediate and construction of a detailed kinetic scheme as shown above for dihydrofolate reductase.
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