Thermodynamic and structural adaptation differences between mesophilic and psychrophilic lactate dehydrogenases, S. Khrapunov, E. Chang and R. H. Callender, Biochemistry 56, 3587-3595 (2017). PMC5574168
In order to further our understanding of protein adaptation, the thermodynamics of substrate binding and enzymatic activity of a glycolytic enzyme, lactate dehydrogenase (LDH), from both porcine heart, phLDH (Sus scrofa; a mespohile), and from mackerel icefish, cgLDH (Chamapsocephalus gunnari; a psychrophile), were investigated. Using a novel and quite sensitive fluorescence assay which can distinguish protein conformational changes close and distal from the substrate binding pocket, a reversible global protein structural transition preceding the high-temperature transition (denaturation) and coinciding with a marked change in enzymatic activity was found for both LDHs. A similar reversible structural transition of active site structure was observed for phLDH but not for cgLDH. An observed lower substrate binding affinity for cgLDH compared to phLDH was accompanied by a higher contribution of entropy to ∆G which reflects a higher functional plasticity of the psychrophilic cgLDH compared to the mesophilic phLDH. A natural osmolyte, TMAO, increases stability and shifts all structural transitions to higher temperatures for both orthologs while simultaneously reducing catalytic activity. The presence of TMAO leads to catalytic parameters for cgLDH like those for phLDH in the absence of the osmolyte. Our results are most naturally understood within a model of enzyme dynamics whereby different conformations of the enzyme interconvert among themselves having varying catalytic parameters (i.e., binding and catalytic proclivity) and population profiles that are both temperature dependent and influenced by osmolytes. Our results also show that adaptation can be overcome by means other than gene mutations and complements the synchronic evolution of the cellular milieu.
Resolution of sub-millisecond kinetics of multiple reaction pathways for lactate dehydrogenase , Michael Reddish, R. H. Callender, and R. B. Dyer, Biophysical J., 112, 1852-1862 (2017). PMC5425397
Enzymes are known to exhibit conformational flexibility. An important consequence of this flexibility is that the same enzyme reaction can occur via multiple reaction pathways on a reaction landscape. A model enzyme for the study of reaction landscapes is lactate dehydrogenase. We have previously used temperature-jump (T-jump) methods to demonstrate that the reaction landscape of lactate dehydrogenase branches at multiple points creating pathways with varied reactivity. A limitation of this previous work is that the T-jump method makes only small perturbations to equilibrium and may not report conclusively on all steps in a reaction. Therefore, interpreting T-jump results of lactate dehydrogenase kinetics has required extensive computational modeling work. Rapid mixing methods offer a complementary approach that can access large perturbations from equilibrium; however, traditional enzyme mixing methods like stopped-flow do not allow for the observation of fast protein dynamics. In this report, we apply a microfluidic rapid mixing device with a mixing time of less than 100 μs that allows us to study these fast dynamics and the catalytic redox step of the enzyme reaction. Additionally, we report UV absorbance and emission T-jump results with improved signal-to-noise at fast times. The combination of mixing and T-jump results yields an unprecedented view of lactate dehydrogenase enzymology confirming the timescale of substrate-induced conformational change and presence of multiple reaction pathways.
The Dynamical Nature of Enzymatic Catalysis, R. Brian Dyer and Robert Callender, Accounts of Chemical Research, 48, 407-413 (2015). PMC4333057
As is well known, enzymes are proteins designed to accelerate specific life essential chemical reactions by many orders of magnitude. A folded protein is a highly dynamical entity, best described as a hierarchy or ensemble of interconverting conformations on all time scales from femtoseconds to minutes. We are just beginning to learn what role these dynamics play in the mechanism of the chemical catalysis by enzymes due to extraordinary difficulties in characterizing the conformational space, i.e. the energy landscape, of a folded protein. It seems clear now that their role is crucially important. Here we discuss approaches, based on vibrational spectroscopies of various sorts, that can reveal the energy landscape of an enzyme-substrate (Michaelis) complex and decipher which part of the typically very complicated landscape is relevant to catalysis. Vibrational spectroscopy is quite sensitive to small changes in bond order and bond length, with a resolution of 0.01 Å or less. It is this sensitivity that is crucial to its ability to discern bond reactivity. Using isotope edited IR approaches, we have studied in detail the role of conformational heterogeneity and dynamics in the catalysis of hydride transfer by LDH (lactate dehydrogenase). Upon the binding of substrate, the LDH-substrate system undergoes a search through conformational space to find a range of reactive conformations over the microsecond to millisecond time scale. The ligand is shuttled to the active site via first forming a weakly bound enzyme-ligand complex, probably consisting of several heterogeneous structures. This complex undergoes numerous conformational changes spread throughout the protein that shuttle the enzyme-substrate complex to a range of conformations where the substrate is tightly bound. This ensemble of conformations all have a propensity towards chemistry but some are much more facile for carrying out chemistry than others. The search for these tightly bound states is clearly directed by the forces that the protein can bring to bear, very much akin to the folding process to form native protein in the first place. In fact, the conformational subspace of reactive conformations of the Michaelis complex can be described as a collapse of reactive sub-states compared to that found in solution, towards a much smaller and much more reactive set. These studies reveal how dynamic disorder in the protein structure can modulate the on-enzyme reactivity. It is very difficult to account for how the dynamical nature of the ground state of the Michaelis complex modulates function by transition state concepts since dynamical disorder is not a starting feature of the theory. We find that dynamical disorder may well play a larger or similar sized role in the measured Gibbs free energy of a reaction compared to the actual energy barrier involved in the chemical event. Our findings are broadly compatible with qualitative concepts of evolutionary adaptation of function such as adaptation to varying thermal environments. Our work suggests a methodology to determine the important dynamics of the Michaelis complex.
Mechanism of Thermal Adaptation in the Lactate Dehydrogenases, Huo-Lei Peng, Tsuyoshi Egawa, Eric Chang, Hua Deng, and Robert Callender, J. Phys. Chem. B, 119, 15256-15262 (2015). PMC4679558
The mechanism of thermal adaptation of enzyme function at the molecular level is poorly understood but is thought to lie within the structure of the protein or its dynamics. Our previous work on pig heart lactate dehydrogenase (phLDH) has determined very high resolution structures of the active site, via isotope edited IR studies, and characterized its dynamical nature, via laser induced temperature jump (T-jump) relaxation spectroscopy on the Michaelis complex. These particular probes are quite powerful at getting at the interplay between structure and dynamics in adaptation. Hence, we extend these studies to the psychrophilic protein cgLDH (Champsocephalus gunnari; 0 degrees C) and the extreme thermophile, tmLDH (Thermotoga maritima LDH; 80 degrees C) for comparison to the mesophile, phLDH (37 degrees C). Instead of the native substrate pyruvate, we utilize oxamate as a non-reactive substrate mimic for experimental reasons. Using isotope edited IR spectroscopy, we find small differences in the sub-state composition that arise from the detailed bonding patterns of oxamate within the active site of the three proteins; however, we find these differences insufficient to explain the mechanism of thermal adaptation. On the other hand, T-jump studies of NADH emission reveal that the most important parameter affecting thermal adaptation appears to be enzyme control of the specific kinetics and dynamics of protein motions that lie along the catalytic pathway. The relaxation rate of the motions scale as cgLDH > phLDH > tmLDH in a way that faithfully matches kcat of the three isozymes.
The Energy Landscape of the Michaelis Complex of Lactate Dehydrogenase: Relationship to Catalytic Mechanism, Huo-Lei Peng, Hua Deng,R. Brian Dyer, and Robert Callender, Biochemistry, 53, 1849-1857 (2014). PMC3985751
Lactate dehydrogenase (LDH) catalyzes the inter-conversion between pyruvate and lactate with NAD as a cofactor. Using isotope edited difference FTIR spectroscopy on the ‘live’ reaction mixture (LDH/NADH/pyruvate <-> LDH/NAD+/lactate) for the wild type protein and a mutant of impaired catalytic efficiency, a set of inter-converting conformational sub-states within the pyruvate side of the Michaelis complex tied to chemical activity is revealed. The important structural features of these sub-states include (1) electronic orbital overlap between pyruvate’s C2=O bond and the nicotinamide ring of NADH, as shown from the observation of a delocalized vibrational mode involving motions from both moieties and (2) a characteristic hydrogen bond distance between pyruvate C2=O and active site residues, as shown by the observation of at least four C2=O stretch bands indicating varying degrees of C2=O bond polarization. These structural features form a critical part of the expected reaction coordinate along the reaction path, and the ability to quantitatively determine them as well as the sub-state population ratios in the Michaelis complex provides a unique opportunity to probe the structure-activity relationship in LDH catalysis. The various substates have a strong variance in their propensity towards on enzyme chemistry. Our results suggest a physical mechanism to understand LDH catalyzed chemistry in which the bulk of the rate enhancement can be viewed as arising from a stochastic search through an available phase space that, in the enzyme system, involves a restricted ensemble of more reactive conformational sub-states as compared to the same chemistry in solution.
Large Scale Dynamics of the Michaelis Complex of B. Stearothermophilus Lactate Dehydrogenase revealed by Single Tryptophan Mutants Study, Beining Nie, Hua Deng, Ruel Desamero, Robert Callender, Biochemistry 52, 1886-1892 (2013). PMC3604157
Large scale dynamics within the Michaelis complex mimic of Bacillus stearothermophilus thermophylic lactate dehydrogenase, bsLDH/NADH/oxamate, were studied with site specific resolution by laser induced temperature jump relaxation spectroscopy having a time resolution of 20 ns. NADH emission and Trp emission from the wild type and a series of single-tryptophan bsLDH mutants, with the tryptophan positions at different distances from the active site, were used as reporters of evolving structure in response to the rapid change in temperature. Several distinct dynamical events were observed on the microsecond - millisecond time-scale involving motion of atoms spread over the protein, some occurring concomitantly or nearly concomitantly with structural changes at the active site. This suggests that a large portion of the protein-substrate complex moves in a rather concerted fashion to bring about catalysis. The catalytically important surface loop undergoes two distinct movements, both needed for a competent enzyme. Our results also suggest that what is called ‘loop motion’ is not just localized to the loop and active site residues. Rather, it involves the motion of atoms spread over the protein, even some quite distal from the active site. How these results bear on catalytic mechanism of bsLDH is discussed.