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Active-Site Heterogeneity of Lactate Dehydrogenase He Yin, Hui Li, Adam Grofe, and Jiali Gao ACS Catal., Just Accepted Manuscript • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 1, 2019
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ACS Catalysis
Active-Site Heterogeneity of Lactate Dehydrogenase
He Yin1, Hui Li1, Adam Grofe1*, Jiali Gao2,3,4*
1. Laboratory of Theoretical and Computational Chemistry, Institute of Theoretical Chemistry, Jilin University, Changchun 130023, P. R. China 2. Department of Chemistry and Supercomputing Institute, University of Minnesota, Minneapolis, Minnesota 55455, United States 3. Lab of Computational Chemistry and Drug Design,
State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen 518055, China 4. Shenzhen Bay Laboratory, Shenzhen 518055, China *Emails: AG:
[email protected]; JG:
[email protected] ABSTRACT: Molecular dynamics simulation of human heart lactate dehydrogenase (LDH) has been carried out to determine the linear and two-dimensional Fourier transform infrared (2D-FTIR) spectra for the carbonyl stretch vibration of pyruvate in the tetrameric enzyme, using quantum vibrational perturbation theory. The computed line-shapes of individual subunits are inhomogeneously broadened, and span the entire absorption range of the carbonyl vibration of the full enzyme, indicating the
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same conformation heterogeneity in the four active sites of LDH. However, each subunit line-shape has different width and peak maximum due to variations in conformation equilibrium in different subunits, corresponding to the spectral multiplets observed experimentally. Since there is a finite time interval before a substrate is converted into products in a given active site, the distribution of such a time coarse-grained average of Michaelis complexes is called active-site heterogeneity. Active-site heterogeneity is distinguished from conformation heterogeneity in that although the former is governed by the same energy landscape that gives rise to conformation heterogeneity, a stochastic enzyme-substrate adduct can only sample a particular fraction of the conformation space, limited by the enzyme turn-over and shown as a distribution of waiting times, i.e., reaction rates, in single-enzyme experiments. The present study showed that different absorption peaks in the C=O stretch region of the Michaelis complex, observed experimentally and reproduced computationally, are due to active-site heterogeneity, as a superposition of the spectral line-shapes of different active sites. Consequently, substates corresponding to these spectral peaks of LDH do not interconvert and they have different reaction rates, as found experimentally. The present active-site heterogeneity mechanism is in complete agreement with the kinetic model derived from isotopeedited infrared and temperature-jump relaxation spectroscopy.
Keywords: Active-site Heterogeneity, Conformation Heterogeneity, Vibrational Frequency of Pyruvate, Lactate Dehydrogenase, Active-site Dynamics
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ACS Catalysis
1. INTRODUCTION Enzymes are dynamic molecules whose fluctuations are essential for function.1-3 As complex as enzymes, the Michaelis-Menten mechanism provides a remarkably satisfactory description of enzymatic kinetics, even at the single-molecule level.4-5 According to this mechanism, all enzyme-catalyzed reactions start from the Michaelis complex, which consists of an ensemble of fluctuating and interconverting conformations on multitude of timescales. If the chemical reaction was slow to allow sufficient sampling of all conformations of the Michaelis complex, there would be only one representative average for a given active site, corresponding to a single minimum on the free energy surface. However, enzymatic reactions are fast; before the substrate is converted into products, just a small fraction of the conformations of the Michaelis complex has been explored, giving rise to different effective averages from stochastic encounters of an enzyme molecule with its substrate, thereby, different observed reaction rates as illustrated by single-molecule experiments,6 including the reaction catalyzed by lactate dehydrogenase (LDH).7 Conformation heterogeneity of the Michaelis complex is governed by its energy landscape,8 which ultimately determines the thermal activation and catalytic rate. Here, we use the term active-site heterogeneity of Michaelis complex to specify an ensemble of the effective average configurations of the active site, sampled in the time interval between substrate binding and barrier crossing. It is distinguished from conformation heterogeneity, which describes the distribution of conformations during that time interval. Conformation heterogeneity gives different effective average configurations of the active site, whereas active-site
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heterogeneity corresponds to different rates of a single enzyme. Further, we use “effective average” to indicate that it is a time coarse-grained average, not a complete ensemble average due to the limited time interval before chemistry takes place in the active site. In the long-time limit, the two terms will be identical for a single enzyme. Alternatively, for an ensemble of molecules, there is no difference between the two terms, giving rise to the phenomenological kcat in the Michaelis-Menten mechanism; however, for each single-enzyme reaction, the effective average configuration and reaction rate would be different.4-5 In this study, we carried out molecular dynamics simulations
to
characterize
conformational
distributions,
i.e.,
conformation
heterogeneity, of protein-substrate interactions in the four active sites of the enzyme LDH, providing insights on the origin of the observed active-site heterogeneity from spectroscopic experiments.9-11 Lactate dehydrogenase is a key metabolic enzyme that catalyzes the interconversion of pyruvate and lactate, involving a hydride transfer from NADH to the substrate pyruvate and a concerted proton transfer from a specific acid residue histidine to the C2 carbonyl oxygen (in the direction of lactate formation). The reaction catalyzed by LDH has been the basis for testing lactate level in the blood stream for conditions ranging from physical exercises to cancer screening, and the reaction mechanism has been extensively investigated and well-characterized.9, 12-15 Callender and coworkers found that polarization of the carbonyl group of pyruvate in the active site can contribute as much as 106 to rate enhancement in LDH catalysis.13, 16 Since specific hydrogen-bonding and electrostatic polarization of the carbonyl group lowers its bond
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ACS Catalysis
order, shown as red-shifted C2=O stretch vibrational frequency, an empirical correlation between measured kcat and the carbonyl frequency shift relative to the solutionphase value has been established following a series studies of the wild-type and mutant LDH enzymes.
kcat ( s 1 ) 100.157( ) 2.5 However, unlike the spectrum of pyruvate in water with a single heterogeneously broadened band,9 the observed vibrational spectra for the substrate often exhibit multiple peaks in the LDH•NADH•Pyruvate complex,9-10 which, on one hand, does not provide a unique value of to assess mechanism, but, on the other hand, is a direct indication of active-site heterogeneity in LDH catalysis.11 An interpretation of the origin and possible interconversion of different substates, responsible for the observed absorption multiplets,9-11, 13-14 could be useful for constructing an accurate model for enzyme catalysis, including the effect of “free-energy” landscape in Michaelis complex. Recently, using isotope-edited FTIR and temperature(T)-jump relaxation spectroscopy, Callender, Dyer and coworkers identified conformational substates that branch into pathways with different reactivity in the Michaelis complex of pig-heart LDH (Scheme 1).9-10 Porcine LDH, as human heart LDH, forms a homotetramer (H4) without cooperativity among the subunits. The reaction mechanism follows a strict order: NADH binding first, followed by substrate, and then, on-enzyme reaction.17 Substrate binding triggers the closure of a small loop (residues 98-110, human heart LDH numbering throughout), encapsulating and bringing a key residue Arg106 into the active site to stabilize the substrate.12, 18-19 Coupled with a newly developed, ultrafast
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mixing technique (
