Protein Mass Effects on Formate Dehydrogenase

Abstract

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 timescale 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 timescales, i.e., 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.

Uncovering the Relationship between the Change in Heat Capacity for Enzyme Catalysis and Vibrational Frequency through Isotope Effect Studies

“…28,37,38 Ranasinghe et al have recently extended this rationale by suggesting that mass modulation not only affects protein motions coupled to the enzyme catalyzed chemical step, but also the electrostatics associated with longer timescale events during turnover. 39 We note that these works have not considered Δ ‡ . Moreover, there have been a significant number of studies that suggest that protein 'dynamics' do not affect enzyme catalysis 17,[40][41][42][43] and are not in any way coupled to the reaction coordinate.…”

Protein Mass-Modulated Effects in Alkaline Phosphatase

“…However, this phenomenon is not present in the AP-catalyzed reaction. Similar examples include the Ec DHFR, Tm DHFR, and FDH proteins …”

“…Enzymes display a hierarchy of motions on varying time scales, which range from seconds to femtoseconds. The role of slower time scale motions has been characterized by both experimental and computational methods, but local, fast catalytic site motions that influence the femtosecond lifetime of the transition states have been limited to computational regimes and require additional experimental investigation for a better understanding of enzyme catalysis. − Experimental approaches that have been useful for unraveling the involvement of slower protein motions in catalysis and some aspects of transition-state properties include nuclear magnetic resonance spectroscopy (NMR), , vibrational spectroscopy, , and the temperature dependence of kinetic isotope effects (KIEs). − A recently developed tool designed to experimentally perturb femtosecond to picosecond time scale fast protein motions involves the labeling of proteins with heavy isotopes ( 13 C, 15 N, and 2 H), uniformly , or by specific amino acids at the catalytic site. − Covalent bond vibrations occur on the femtosecond time scale, and isotopic substitutions have immediate consequences on this time scale. However, heavy protein effects are certainly propagated throughout the protein, and effects on slower steps, including substrate binding, conformational changes, and product release, are also possible.…”

Heavy Enzymes and the Rational Redesign of Protein Catalysts

“…Strategies previously employed in the literature by various groups include isotopically labelling the entire enzyme or labelling either of single amino acid residues or of particular segments, such as mobile loops (Figure ) . The effects of protein isotope labelling on transition states have been characterised in different enzymes including purine nucleotide phosphorylase (PNP), HIV protease (HIV‐1 PR), alanine racemase, dihydrofolate reductase (DHFR), pentaerythritol tetranitrate reductase (PETNR), formate dehydrogenase (FDH), lactate dehydrogenase (LDH) and alcohol dehydrogenase . In most of these cases, isotope labelling reduced the rate of the chemical step ––.…”