The power of integrating kinetic isotope effects into the formalism of the Michaelis-Menten equation

Abstract

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.

Keywords: enzymatic C-H activation; kinetic isotope effects; mechanism of enzyme action; mechanism of two copper monooxygenases; steady-state kinetics.

Figure 1

X-ray structure of oxidized PHM. Coppers at the Cu_M and Cu_H sites are in the +2 oxidation state. The substrate analog, Ac-DiI-YG [acyl-diiodo-Tyr-Gly] is in stick form. The conserved ligands at Cu_M [H242, H244, and Met314] and at Cu_H [H107, H108, and H172] are in green. The conserved tyrosines are yellow [Y318 and Y79] (13, 14). Reprinted with permission from the Journal of the American Chemical Society, Vol. 126, pp. 13168–13169. © 2004 American Chemical Society.

Figure 2

The size of the tritium isotope as a function of O₂ levels in the DβM reaction. The y axis, V/K)_H/V/K)_T is ^T(k_cat/K_m) in eq [4] of the text (23). Reprinted with permission from the Journal of Biological Chemistry, Vol. 255, pp. 11648–11651. ©1980 The American Society for Biochemistry and Molecular Biology.

Figure 3

Temperature dependence of the intrinsic deuterium isotope effect on k_(C–H) for PHM (32). Reprinted with permission from the Journal of the American Chemical Society, Vol. 124, pp. 8194–8195. © 2002 American Chemical Society.

Figure 4

Marcus-like model of H-tunneling (53). The position of the heavy atoms is shown in (a) and the position of the H-atom is in (b). The movement of the heavy atoms generates a tunneling-ready state that allows the H-wave function to be delocalized between the reactant and product wells [middle graphs in (a) and (b)]. When the donor-acceptor distance is non-optimal, a mass- and temperature-dependent sampling of the donor-acceptor distance enters into the reaction coordinate. This is illustrated in (c), and represents the origin of the temperature-dependent KIEs. Reprinted with permission from the Annual Reviews of Biochemistry, Vol. 82. © 2013 Annual Reviews.

Scheme 1

The C–H activation step of DβM, TβM, and PHM is proposed to occur via a hydrogen atom transfer to an adjacent Cu_M(O₂·⁻). Though the latter has never been experimentally detected, all mechanistic probes converge on the illustrated, “consensus” mechanism (15).