Examination of enzymatic H-tunneling through kinetics and dynamics

Abstract

In recent years, kinetic measurements of isotope effects of enzyme-catalyzed reactions and their temperature dependence led to the development of theoretical models that were used to rationalize the findings. These models suggested that motions at the femto- to picosecond (fs to ps) time scale modulate the environment of the catalyzed reaction. Due to the fast nature of motions that directly affect the cleavage of a covalent bond, it is challenging to correlate the enzyme kinetics and dynamics related to that step. We report a study of formate dehydrogenase (FDH) that compares the temperature dependence of intrinsic kinetic isotope effects (KIEs) to measurements of the environmental dynamics at the fs-ps time scale (Bandaria et al. J. Am. Chem. Soc. 2008, 130, 22-23). The findings from this comparison of experimental kinetics and dynamics are consistent with models of environmentally coupled H-tunneling models, also known as Marcus-like models. Apparently, at tunneling ready conformations, the donor-acceptor distance, orientation, and fluctuations seems to be well tuned for H-transfer and are not affected by thermal fluctuations slower than 10 ps. This phenomenon has been suggested in the past to be quite general in enzymatic reactions. Here, the kinetics and the dynamics measurements on a single chemical step and on fs-ps time scale, respectively, provide new insight and support for the relevant theoretical models. Furthermore, this methodology could be applied to other systems and be used to examine mutants for which the organization of the donor and acceptor is not ideal, or enzymes with different rigidity and different temperature optimum.

Figure 1

An illustration of a Marcus-like model. The “Marcus-term” axis indicates motions that carry the system to the TRC (‡). The “gating” axis indicates the fluctuations of the DAD. R and P are the reactant and product potential energy surfaces, respectively. The red curves show the wavefunctions of the hydrogen nucleus. Reproduced with permission from Willy.

Figure 2

The reaction catalyzed by FDH, with an illustration of the reaction’s TRC or transition state (‡). Below the TRC the relative orientation of the TSA azide, bent as observed in the crystal structure (Figure 3), and the substrate NAD+ is presented. It is apparent that the charge distribution of the azide and the TRC are similar, in accordance with its identification as TSA.,

Figure 3

Active site structure of FDH-azide-NAD+ complex (PDB # 2NAD)., All the nitrogens are in blue and the NAD is in magenta. The arrow indicates the reaction path from the H- donor to acceptor and the dashed lines represent the hydrogen bonds discussed in the text. All distances are in Å.

Figure 4

An Arrhenius plot of the intrinsic H/T (red), H/D (blue) and D/T (green) KIEs (log scale) vs. the reciprocal of the absolute temperature. The average KIEs are presented as points and the lines are an exponential fit of all the data points to the Arrhenius equation.

Figure 5

A plot of the FFCF (C(t)) for the FDH-azide- NAD+. Data are from ref and the graph presents the decay of the function as function of T_W (see text).