RECENT HIGHLIGHTS OF CURRENT RESEARCH
Triple Isotope Effects Support Concerted Hydride and Proton Transfer and Promoting Vibrations in Human Heart Lactate Dehydrogenase , Zhen Wang, Eric P. Chang, and Vern L. Schramm, J. Am. Chem. Soc. 138, 15004-15010 (2016). pdf
Transition path sampling simulations have proposed that human heart lactate dehydrogenase (LDH) employs protein promoting vibrations (PPVs) on the femto- second (fs) to picosecond (ps) time scale to promote crossing of the chemical barrier. This chemical barrier involves both hydride and proton transfers to pyruvate to form L-lactate, using reduced nicotinamide adenine dinucleotide (NADH) as the cofactor. Here we report experimental evidence from three types of isotope effect experiments that support coupling of the promoting vibrations to barrier crossing and the coincidence of hydride and proton transfer. We prepared the native (light) LDH and a heavy LDH labeled with 13C, 15N, and nonexchangeable 2H (D) to perturb the predicted PPVs. Heavy LDH has slowed chemistry in single turnover experiments, supporting a contribution of PPVs to transition state formation. Both the [4-2H]NADH (NADD) kinetic isotope effect and the D2O solvent isotope effect were increased in dual-label experiments combining both NADD and D2O, a pattern maintained with both light and heavy LDHs. These isotope effects support concerted hydride and proton transfer for both light and heavy LDHs. Although the transition state barrier-crossing probability is reduced in heavy LDH, the concerted mechanism of the hydride−proton transfer reaction is not altered. This study takes advantage of triple isotope effects to resolve the chemical mechanism of LDH and establish the coupling of fs-ps protein dynamics to barrier crossing.
Targeting a Rate-Promoting Vibration with an Allosteric Mediator in Lactate Dehydrogenase , Michael W. Dzierlenga and Steven D. Schwartz, J. Phys. Chem. Letts 7, 2591-2596 (2016). pdf
We present a new type of allosteric modulation in which a molecule bound outside the active site modifies the chemistry of an enzymatic reaction through rapid protein dynamics. As a test case for this type of allostery, we chose an enzyme with a well-characterized rate-promoting vibration, lactate dehydrogenase; identified a suitable small molecule for binding; and used transition path sampling to obtain ensembles of reactive trajectories. We found that the small molecule significantly affected the reaction by changing the position of the transition state and, through applying committor distribution analysis, showed that it removed the protein component from the reaction coordinate. The ability of a small-molecule to disrupt enzymatic reactions through alteration of subpicosecond protein motion opens the door for new experimental studies on protein motion coupled to enzymatic reactions and possibly the design of drugs to target these enzymes.
Mechanisms of Thermal Adaptation in the Lactate Dehydrogenases, H.-L. Peng, T. Egawa, E. Chang, H. Deng and R. H. 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 °C) and the extreme thermophile, tmLDH (Thermotoga maritima LDH; 80 °C) for comparison to the mesophile, phLDH (37 °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 Dynamical Nature of Enzymatic Catalysis, R. Callender and R. B. Dyer, 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 0.01Å 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.