The power of integrating kinetic isotope effects into the formalism of the Michaelis-Menten equation.
The final arbiter of enzyme mechanism is the ability to establish and test a kinetic mechanism. Isotope effects play a major role in expanding the scope and insight derived from the Michaelis-Menten equation. The integration of isotope effects into the formalism of the Michaelis-Menten equation began in the 1970s and has continued until the present. This review discusses a family of eukaryotic copper proteins, including dopamine β-monooxygenase, tyramine β-monooxygenase and peptidylglycine α-amidating enzyme, which are responsible for the synthesis of neuroactive compounds, norepinephrine, octopamine and C-terminally carboxamidated peptides, respectively. The review highlights the results of studies showing how combining kinetic isotope effects with initial rate parameters permits the evaluation of: (a) the order of substrate binding to multisubstrate enzymes; (b) the magnitude of individual rate constants in complex, multistep reactions; (c) the identification of chemical intermediates; and (d) the role of nonclassical (tunnelling) behaviour in C-H activation.
Departments of Chemistry and Molecular and Cell Biology, California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA.
Judith P
JP
Klinman
D000818:Animals; D055162:Biocatalysis; D020134:Catalytic Domain; D004299:Dopamine beta-Hydroxylase / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry; D011485:Protein Binding; D014316:Tritium / Q000737:chemistry
D009097:Multienzyme Complexes; D014316:Tritium; D006899:Mixed Function Oxygenases; D004299:Dopamine beta-Hydroxylase; C035189:peptidylglycine monooxygenase
enzymatic C-H activation; kinetic isotope effects; mechanism of enzyme action; mechanism of two copper monooxygenases; steady-state kinetics