Kinetic isotope effects, elucidating reaction mechanisms with

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 cw-enediol intermediate and construction of a detailed kinetic scheme as shown above for dihydrofolate reductase. 

In the last ten years the exploration of the kinetics and the mechanism of these reactions has become very fruitful, owing to the continuous effort of research workers in different countries all over the world, who made use of all the modern tools now available in catalytic research. Especially the simultaneous application of different measuring techniques, for instance the infrared study of adsorbed reaction intermediates combined with the study of kinetics and kinetic isotope effects, appeared to be very elucidating. 

Studies of kinetic-isotope effects have also provided valuable information about the mechanisms of reactions, and have been very helpful in elucidating biological mechanisms. Unfortunately, thje theory is somewhat complicated, and there are a few pitfalls. Only a very general and brief outline can be given here. For further details, with special reference to biological mechanisms, the reader is referred to the book by Laidler and Bunting listed in Selected Reading at the end of this chapter. 

Apart from kinetic measurements, the studies of oxygen adsorption on silver (63, 64), electron work function variations accompanying oxygen adsorption (65-67), heat effects of adsorption (65), reactivity of oxygen adsorbed on silver (67), and oxygen isotopic exchange on silver (56-55) were used for the elucidation of the mechanism of the reaction. The papers cited contain references to the works of other authors that were used for the formulation of the reaction mechanism on a level with our results. Here we shall mention, first of all, the work by Twigg (69), who has ascertained... 

The role of the metal ion in ester hydrolysis catalysed by CPA has been examined with both Zn +- and Co +-substituted enzymes. When the terminal carboxyl of the substrate is electrostatically linked to argenine-145 and the aromatic side-chain lies in a hydrophobic pocket, the only residues close enough to the substrate to enter catalysis are glutamate-270, tyrosine-248, the metal ion, and its associated water. Low-temperature studies aid the elucidation of the mechanism. Between - 25 and - 45 °C in ethylene glycol-water mixtures two kinetically discrete processes are detected, the slower of which corresponds to the catalytic rate constant. The faster reaction is interpreted as deacylation of a mixed anhydride acyl-enzyme intermediate formed by nucleophilic attack by glutamate-270 on the substrate (Scheme 6). Differences in the acidity dependences of the catalytic rate constant with the metal ions Zn + (p STa 6.1) and Co +-(pATa 4.9) suggest that ionization of the metal-bound water molecule occurs and is involved in the decay of the anhydride. The catalytic rate constant shows an isotope effect in DgO. 

---