Enzymes with hydrogen isotopes

Even nowadays the application of radioactive isotopes is the most sensitive method for the analysis of biomolecules or their reaction products. Besides the low detection limits, the replacement of a naturally overbalancing stable isotope by its radioactive analogue does not interfere with the physical or chemical properties of the enzyme (with some exceptions for hydrogens). Figure 6 lists some frequently used radioactive isotopes and their half-life periods. 

OTHER EXAMPLES OF KINETIC ISOTOPE EFFECTS. The power of kinetic isotope effects in enzymol-ogy is well illustrated in the work of Rose ° and Knowles deahng with hydrogen effects in proton transfer to and from carbon. Abstraction of a proton from a tetrahedral carbon is a fundamental step in many enzyme-catalyzed reactions. Intramolecular proton transfer as well as partial loss (wash-out) migrating protons have provided important clues in mechanistic investigations. Enol and enediolate formation constitute several... 

Exact localization of the hydrogen isotope is usually required, and it is inadvisable to rely on the method of synthesis, as the label is not always situated entirely at the position predicted.69 In many mechanistic and biological studies with enzymes, the position of the label alters during the reaction, and, in others, where a proton isotope from the solvent is incorporated, it is important to localize the incorporated isotope, and, if it is at a methylene group, to determine its stereochemistry. 

Small isotope-effects can be detected by double-labeling techniques, in which the carbon skeleton is labeled with 14C, and the ratio of 14C to tritium is measured both in the substrate and the product. Care must be taken in the observation and interpretation of isotope effects determined from the hydrogen-isotope content of the product. Just as in non-enzymic reactions (see p. 154), discrimination against the substrate containing deuterium or tritium leads to an increase in the isotopic content of the substrate, and this decreases the apparent isotope-effect towards the end of the reaction. 

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