Isotopic Labeling of Formate Dehydrogenase Perturbs the Protein Dynamics.

Isotope substitution of enzymes has become a means of addressing the participation of protein motions in enzyme-catalyzed reactions. The idea is that only the enzyme mass will be altered and not the electrostatics, so that the protein dynamics are essentially the same but at lower frequencies because of the mass change. In this study, we variably label all carbon atoms in formate dehydrogenase (FDH) with 13C, all nitrogen atoms with 15N, and all nonexchangeable hydrogen atoms with deuterium and investigate the impact that isotopic substitution has on the dynamics at the active site by two-dimensional infrared spectroscopy and compare with the measurements of the temperature dependence of the intrinsic kinetic isotope effects (KIEs). We show that 15N labeling of FDH has the largest effect and makes the active site more heterogeneous, whereas the addition of nonexchangeable deuterium appears to have the opposite effect of 15N on active-site dynamics, resulting in a behavior similar to that of native FDH. Nevertheless, the temperature dependence of the KIEs shows a monotonic trend with protein mass that does not correspond with the changes in dynamics. These results suggest that isotope labeling has more than just a mass effect on enzyme dynamics and may influence electrostatics in ways that complicate the interpretation of the protein isotope effect.

Oscillatory Active-site Motions Correlate with Kinetic Isotope Effects in Formate Dehydrogenase.

Thermal motions of enzymes have been invoked to explain the temperature dependence of kinetic isotope effects (KIE) in enzyme-catalyzed hydride transfers. Formate dehydrogenase (FDH) from Candida boidinii exhibits a temperature independent KIE that becomes temperature dependent upon mutation of hydrophobic residues in the active site. Ternary complexes of FDH that mimic the transition state structure allow investigation of how these mutations influence active-site dynamics. A combination of X-ray crystallography, two-dimensional infrared (2D IR) spectroscopy, and molecular dynamic simulations characterize the structure and dynamics of the active site. FDH exhibits oscillatory frequency fluctuations on the picosecond timescale, and the amplitude of these fluctuations correlates with the temperature dependence of the KIE. Both the kinetic and dynamic phenomena can be reproduced computationally. These results provide experimental evidence for a connection between the temperature dependence of KIEs and motions of the active site in an enzyme-catalyzed reaction consistent with activated tunneling models of the hydride transfer reaction.

Protein Mass Effects on Formate Dehydrogenase.

Isotopically labeled enzymes (denoted as "heavy" or "Born-Oppenheimer" enzymes) have been used to test the role of protein dynamics in catalysis. The original idea was that the protein's higher mass would reduce the frequency of its normal-modes without altering its electrostatics. Heavy enzymes have been used to test if the vibrations in the native enzyme are coupled to the chemistry it catalyzes, and different studies have resulted in ambiguous findings. Here the temperature-dependence of intrinsic kinetic isotope effects of the enzyme formate dehydrogenase is used to examine the distribution of H-donor to H-acceptor distance as a function of the protein's mass. The protein dynamics are altered in the heavy enzyme to diminish motions that determine the transition state sampling in the native enzyme, in accordance with a Born-Oppenheimer-like effect on bond activation. Findings of this work suggest components related to fast frequencies that can be explained by Born-Oppenheimer enzyme hypothesis (vibrational) and also slower time scale events that are non-Born-Oppenheimer in nature (electrostatic), based on evaluations of protein mass dependence of donor-acceptor distance and forward commitment to catalysis along with steady state and single turnover measurements. Together, the findings suggest that the mass modulation affected both local, fast, protein vibrations associated with the catalyzed chemistry and the protein's macromolecular electrostatics at slower time scales; that is, both Born-Oppenheimer and non-Born-Oppenheimer effects are observed. Comparison to previous studies leads to the conclusion that isotopic labeling of the protein may have different effects on different systems, however, making heavy enzyme studies a very exciting technique for exploring the dynamics link to catalysis in proteins.