Isotope effects from partitioning of intermediates in enzyme-catalyzed hydroxylation reactions

Research output: Chapter in Book/Report/Conference proceedingChapter

10 Citations (Scopus)

Abstract

As discussed elsewhere in this volume, substitution with a heavier isotope of an atom in the substrate for an enzyme-catalyzed reaction will not necessarily result in a detectable change in either the rate of product formation in a single assay or on the Vmax and V/K values determined from more complete kinetic analyses. Expression of intrinsic isotope effects on Vmax values can be masked by slower chemical steps steps which are not isotope-sensitive and by slow product release, while effects on V/K values can be masked by high commitments to catalysis.1 Occasionally, these problems can be minimized by either changing the conditions, e.g., carrying out assays off the pH optimum,2 or by utilizing a slower substrate. However, in the case of enzymes which carry out hydroxylation reactions, it is frequently found that neither of these approaches are successful. The reactions of many hydroxylases can be divided into two discrete chemical steps: the reaction of oxygen with an enzyme bound cofactor such as a metal to form the hydroxylating intermediate and the subsequent reaction of this intermediate with the organic substrate. Formation of the hydroxylating intermediate is generally irreversible; thus, if one is interested in using kinetic isotope effects to probe the mechanism of hydroxylation, V/K isotope effects are ruled out because the isotope-sensitive step occurs after an irreversible step. If formation of the hydroxylating intermediate is much slower than the subsequent hydroxylation or if product release is slow, Vmax isotope effects are significantly smaller than the intrinsic values. This problem cannot necessarily be circumvented by using a slow substrate, in that the hydroxylating intermediate is unstable and will often carry out an alternative reaction if the substrate is too unreactive. This alternative pathway may be simply unproductive breakdown of the hydroxylating intermediate or a reaction at a different site on the substrate. As discussed here, either of these alternative pathways can be used to determine kinetic isotope effects which are not reflected in V/K or Vmax values.

Original languageEnglish (US)
Title of host publicationIsotope Effects in Chemistry and Biology
PublisherCRC Press
Pages861-873
Number of pages13
ISBN (Electronic)9781420028027
ISBN (Print)9780824724498
DOIs
StatePublished - Jan 1 2005
Externally publishedYes

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Hydroxylation
Isotopes
Enzymes
Substrates
Kinetics
Assays
Coenzymes
Mixed Function Oxygenases
Substitution reactions
Metals
Oxygen
Atoms

ASJC Scopus subject areas

  • Chemistry(all)
  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Isotope effects from partitioning of intermediates in enzyme-catalyzed hydroxylation reactions. / Fitzpatrick, Paul F.

Isotope Effects in Chemistry and Biology. CRC Press, 2005. p. 861-873.

Research output: Chapter in Book/Report/Conference proceedingChapter

Fitzpatrick, Paul F. / Isotope effects from partitioning of intermediates in enzyme-catalyzed hydroxylation reactions. Isotope Effects in Chemistry and Biology. CRC Press, 2005. pp. 861-873
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