Co-reporter:Zhen Wang, Paul J. Sapienza, Thelma Abeysinghe, Calvin Luzum, Andrew L. Lee, Janet S. Finer-Moore, Robert M. Stroud, and Amnon Kohen
Journal of the American Chemical Society May 22, 2013 Volume 135(Issue 20) pp:7583-7592
Publication Date(Web):April 23, 2013
DOI:10.1021/ja400761x
Thymidylate synthase (TSase) produces the sole intracellular de novo source of thymidine (i.e., the DNA base T) and thus is a common target for antibiotic and anticancer drugs. Mg2+ has been reported to affect TSase activity, but the mechanism of this interaction has not been investigated. Here we show that Mg2+ binds to the surface of Escherichia coli TSase and affects the kinetics of hydride transfer at the interior active site (16 Å away). Examination of the crystal structures identifies a Mg2+ near the glutamyl moiety of the folate cofactor, providing the first structural evidence for Mg2+ binding to TSase. The kinetics and NMR relaxation experiments suggest that the weak binding of Mg2+ to the protein surface stabilizes the closed conformation of the ternary enzyme complex and reduces the entropy of activation on the hydride transfer step. Mg2+ accelerates the hydride transfer by ∼7-fold but does not affect the magnitude or temperature dependence of the intrinsic kinetic isotope effect. These results suggest that Mg2+ facilitates the protein motions that bring the hydride donor and acceptor together, but it does not change the tunneling ready state of the hydride transfer. These findings highlight how variations in cellular Mg2+ concentration can modulate enzyme activity through long-range interactions in the protein, rather than binding at the active site. The interaction of Mg2+ with the glutamyl tail of the folate cofactor and nonconserved residues of bacterial TSase may assist in designing antifolates with polyglutamyl substitutes as species-specific antibiotic drugs.
Co-reporter:Svetlana A. Kholodar
Journal of the American Chemical Society 2016 Volume 138(Issue 26) pp:8056-8059
Publication Date(Web):June 21, 2016
DOI:10.1021/jacs.6b03826
Thymidylate synthase is an attractive target for antibiotic and anticancer drugs due to its essential role in the de novo biosynthesis of the DNA nucleotide thymine. The enzymatic reaction is initiated by a nucleophilic activation of the substrate via formation of a covalent bond to an active site cysteine. The traditionally accepted mechanism is then followed by a series of covalently bound intermediates, where that bond is only cleaved upon product release. Recent computational and experimental studies suggest that the covalent bond between the protein and substrate is actually quite labile. Importantly, these findings predict the existence of a noncovalently bound bisubstrate intermediate, not previously anticipated, which could be the target of a novel class of drugs inhibiting DNA biosynthesis. Here we report the synthesis of the proposed intermediate and findings supporting its chemical and kinetic competence. These findings substantiate the predicted nontraditional mechanism and the potential of this intermediate as a new drug lead.
Co-reporter:Kevin Francis, Paul J. Sapienza, Andrew L. Lee, and Amnon Kohen
Biochemistry 2016 Volume 55(Issue 7) pp:1100-1106
Publication Date(Web):January 26, 2016
DOI:10.1021/acs.biochem.5b00945
Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C–H → C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs), which are sensitive to the physical nature of the chemical step, and protein mass modulation, which slows down fast dynamics (femto- to picosecond time scale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5 to 45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction’s transition state (or tunneling ready state, TRS). Mass modulation of these enzymes through isotopic labeling with 13C, 15N, and 2H at nonexchangeable hydrogens yields an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass modulation of the human DHFR affects neither DAD distribution nor the DAD’s conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C–H → C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically modulated heavy enzymes in general.
Co-reporter:Qi Guo, Lokesh Gakhar, Kyle Wickersham, Kevin Francis, Alexandra Vardi-Kilshtain, Dan T. Major, Christopher M. Cheatum, and Amnon Kohen
Biochemistry 2016 Volume 55(Issue 19) pp:2760-2771
Publication Date(Web):April 21, 2016
DOI:10.1021/acs.biochem.6b00181
The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for studies on the role of protein dynamics in catalysis, but so far these studies have been limited by the availability of structural information. Second, CbFDH and its mutants can be used in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in commercial development. Here, we report the crystallization and structural determination of both holo- and apo-CbFDH. The free-energy barrier for the catalyzed reaction was computed and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: the apoenzyme crystal was obtained under cocrystallization conditions with a saturating concentration of both the cofactor (NAD+) and inhibitor (azide), which has a nanomolar dissociation constant. It was found that the fraction of the apoenzyme present in the solution is less than 1.7 × 10–7 (i.e., the solution is 99.9999% holoenzyme). This is an extreme case where the crystal structure represents an insignificant fraction of the enzyme in solution, and a mechanism rationalizing this phenomenon is presented.
Co-reporter:Amnon Kohen
Accounts of Chemical Research 2015 Volume 48(Issue 2) pp:466
Publication Date(Web):December 24, 2014
DOI:10.1021/ar500322s
The role of the enzyme’s dynamic motions in catalysis is at the center of heated contemporary debates among both theoreticians and experimentalists. Resolving these apparent disputes is of both intellectual and practical importance: incorporation of enzyme dynamics could be critical for any calculation of enzymatic function and may have profound implications for structure-based drug design and the design of biomimetic catalysts.Analysis of the literature suggests that while part of the dispute may reflect substantial differences between theoretical approaches, much of the debate is semantic. For example, the term “protein dynamics” is often used by some researchers when addressing motions that are in thermal equilibrium with their environment, while other researchers only use this term for nonequilibrium events. The last cases are those in which thermal energy is “stored” in a specific protein mode and “used” for catalysis before it can dissipate to its environment (i.e., “nonstatistical dynamics”). This terminology issue aside, a debate has arisen among theoreticians around the roles of nonstatistical vs statistical dynamics in catalysis. However, the author knows of no experimental findings available today that examined this question in enzyme catalyzed reactions.Another source of perhaps nonsubstantial argument might stem from the varying time scales of enzymatic motions, which range from seconds to femtoseconds. Motions at different time scales play different roles in the many events along the catalytic cascade (reactant binding, reprotonation of reactants, structural rearrangement toward the transition state, product release, etc.). In several cases, when various experimental tools have been used to probe catalytic events at differing time scales, illusory contradictions seem to have emerged. In this Account, recent attempts to sort the merits of those questions are discussed along with possible future directions.A possible summary of current studies could be that enzyme, substrate, and solvent dynamics contribute to enzyme catalyzed reactions in several ways: first via mutual “induced-fit” shifting of their conformational ensemble upon binding; then via thermal search of the conformational space toward the reaction’s transition-state (TS) and the rare event of the barrier crossing toward products, which is likely to be on faster time scales then the first and following events; and finally via the dynamics associated with products release, which are rate-limiting for many enzymatic reactions. From a chemical perspective, close to the TS, enzymatic systems seem to stiffen, restricting motions orthogonal to the chemical coordinate and enabling dynamics along the reaction coordinate to occur selectively. Studies of how enzymes evolved to support those efficient dynamics at various time scales are still in their infancy, and further experiments and calculations are needed to reveal these phenomena in both enzymes and uncatalyzed reactions.
Co-reporter:Priyanka Singh, Kevin Francis, and Amnon Kohen
ACS Catalysis 2015 Volume 5(Issue 5) pp:3067
Publication Date(Web):April 8, 2015
DOI:10.1021/acscatal.5b00331
Molecular dynamics calculations and bionformatic studies of dihydrofolate reductase (DHFR) have suggested a network of coupled motions across the whole protein that is correlated to the reaction coordinate. Experimental studies demonstrated that distal residues G121, M42, and F125 in E. coli DHFR participate in that network. The missing link in our understanding of DHFR catalysis is the lack of a mechanism by which such remote residues can affect the catalyzed chemistry at the active site. Here, we present a study of the temperature dependence of intrinsic kinetic isotope effects (KIEs) that indicates synergism between a remote residue in that dynamic network, G121, and the active site’s residue I14. The intrinsic KIEs for the I14A–G121V double mutant showed steeper temperature dependence (ΔEa(T-H)) than expected from comparison of the wild type and two single mutants. That effect was nonadditive (i.e., ΔEa(T-H) G121V + ΔEa(T-H) I14A < ΔEa(T-H) double mutant), which indicates a synergism between the two residues. This finding links the remote residues in the network under investigation to the enzyme’s active site, providing a mechanism by which these residues can be coupled to the catalyzed chemistry. This experimental evidence validates calculations proposing that both remote and active site residues constitute a network of coupled promoting motions correlated to the bond activation step (C–H → C hydride transfer in this case). Additionally, the effect of I14A and G121V mutations on single turnover rates was additive rather than synergistic. Although single turnover rate measurements are more readily available and thus more popular than assessing intrinsic KIEs, the current finding demonstrates that these rates, which in DHFR reflect several microscopic rate constants, can fall short of revealing the nature of the C–H bond activation per se.Keywords: dihydrofolate reductase; dynamic network; enzyme dynamics; kinetic isotope effects; tunneling
Co-reporter:Zahidul Islam, Timothy S. Strutzenberg, Ananda K. Ghosh, and Amnon Kohen
ACS Catalysis 2015 Volume 5(Issue 10) pp:6061
Publication Date(Web):September 2, 2015
DOI:10.1021/acscatal.5b01332
Thymidylate synthase (TSase) catalyzes the de novo biosynthesis of thymidylate, a precursor for DNA, and is thus an important target for chemotherapeutics and antibiotics. Two sequential C–H bond cleavages catalyzed by TSase are of particular interest: a reversible proton abstraction from the 2′-deoxyuridylate substrate, followed by an irreversible hydride transfer forming the thymidylate product. QM/MM calculations of the former predicted a mechanism in which the abstraction of the proton leads to formation of a novel nucleotide-folate intermediate that is not covalently bound to the enzyme (Wang, Z.; Ferrer, S.; Moliner, V.; Kohen, A. Biochemistry 2013, 52, 2348–2358). Existence of such an intermediate would hold promise as a target for a new class of drugs. Calculations of the subsequent hydride transfer predicted a concerted H transfer and elimination of the enzymatic cysteine (Kanaan, N.; Ferrer, S.; Marti, S.; Garcia-Viloca, M.; Kohen, A.; Moliner, V. J. Am. Chem. Soc. 2011, 133, 6692–6702). A key to both C–H activations is a highly conserved arginine (R166) that stabilizes the transition state of both H transfers. Here, we test these predictions by studying the R166-to-lysine mutant of Escherichia coli TSase (R166 K) using intrinsic kinetic isotope effects and their temperature dependence to assess effects of the mutation on both chemical steps. The findings confirmed the predictions made by the QM/MM calculations, implicate R166 as an integral component of both reaction coordinates, and thus provide critical support to the nucleotide-folate intermediate as a new target for rational drug design.Keywords: C−H bond activation; donor and acceptor distances; kinetic isotope effect; phenomenological models; QM/MM calculations; thymidylate synthase; tunneling ready state
Co-reporter:Ananda K. Ghosh, Zahidul Islam, Jonathan Krueger, Thelma Abeysinghe and Amnon Kohen
Physical Chemistry Chemical Physics 2015 vol. 17(Issue 46) pp:30867-30875
Publication Date(Web):15 Apr 2015
DOI:10.1039/C5CP01246E
The enzyme thymidylate synthase (TSase), an important chemotherapeutic drug target, catalyzes the formation of 2′-deoxythymidine-5′-monophosphate (dTMP), a precursor of one of the DNA building blocks. TSase catalyzes a multi-step mechanism that includes the abstraction of a proton from the C5 of the substrate 2′-deoxyuridine-5′-monophosphate (dUMP). Previous studies on ecTSase proposed that an active-site residue, Y94 serves the role of the general base abstracting this proton. However, since Y94 is neither very basic, nor connected to basic residues, nor located close enough to the pyrimidine proton to be abstracted, the actual identity of this base remains enigmatic. Based on crystal structures, an alternative hypothesis is that the nearest potential proton-acceptor of C5 of dUMP is a water molecule that is part of a hydrogen bond (H-bond) network comprised of several water molecules and several protein residues including H147, E58, N177, and Y94. Here, we examine the role of the residue Y94 in the proton abstraction step by removing its hydroxyl group (Y94F mutant). We investigated the effect of the mutation on the temperature dependence of intrinsic kinetic isotope effects (KIEs) and found that these KIEs are more temperature dependent than those of the wild-type enzyme (WT). These results suggest that the phenolic –OH of Y94 is a component of the transition state for the proton abstraction step. The findings further support the hypothesis that no single functional group is the general base, but a network of bases and hydroxyls (from water molecules and tyrosine) sharing H-bonds across the active site can serve the role of the general base to remove the pyrimidine proton.
Co-reporter:Thushani D. Nilaweera, Muhammad Saeed, and Amnon Kohen
Biochemistry 2015 Volume 54(Issue 5) pp:1287-1293
Publication Date(Web):January 12, 2015
DOI:10.1021/bi501481n
The development of cancer-specific probes for imaging by positron emission tomography (PET) is gaining impetus in cancer research and clinical oncology. One of the hallmarks of most cancer cells is incessant DNA replication, which requires the continuous synthesis of nucleotides. Thymidylate synthase (TSase) is unique in this context because it is the only enzyme in humans that is responsible for the de novo biosynthesis of the DNA building block 2′-deoxy-thymidylate (dTMP). TSase catalyzes the reductive methylation of 2′-deoxy-uridylate (dUMP) to dTMP using (R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (MTHF) as a cofactor. Not surprisingly, several human cancers overexpress TSase, which makes it a common target for chemotherapy (e.g., 5-fluorouracil). We envisioned that [11C]-MTHF might be a PET probe that could specifically label cancerous cells. Using stable radiotracer [14C]-MTHF, we had initially found increased uptake by breast and colon cancer cell lines. In the current study, we examined the uptake of this radiotracer in human pancreatic cancer cell lines MIAPaCa-2 and PANC-1 and found predominant radiolabeling of cancerous versus normal pancreatic cells. Furthermore, uptake of the radiotracer is dependent on the intracellular level of the folate pool, cell cycle phase, expression of folate receptors on the cell membrane, and cotreatment with the common chemotherapeutic drug methotrexate (MTX, which blocks the biosynthesis of endogenous MTHF). These results point toward [11C]-MTHF being used as PET probe with broad specificity and being able to control its signal through MTX co-administration.
Co-reporter:Tatiana V. Mishanina
Journal of Labelled Compounds and Radiopharmaceuticals 2015 Volume 58( Issue 9) pp:370-375
Publication Date(Web):
DOI:10.1002/jlcr.3313
Flavin nucleotides, i.e. flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are utilized as prosthetic groups and/or substrates by a myriad of proteins, ranging from metabolic enzymes to light receptors. Isotopically labeled flavins have served as invaluable tools in probing the structure and function of these flavoproteins. Here we present an enzymatic synthesis of several radio- and stable-isotope labeled flavin nucleotides from commercially available labeled riboflavin and ATP. The synthetic procedure employs a bifunctional enzyme, Corynebacterium ammoniagenes FAD synthetase, that sequentially converts riboflavin to FMN and then to FAD. The final flavin product (FMN or FAD) is controlled by the concentration of ATP in the reaction. Utility of the synthesized labeled FAD cofactors is demonstrated in flavin-dependent thymidylate synthase. The described synthetic approach can be easily applied to the production of flavin nucleotide analogues from riboflavin precursors.
Co-reporter:Priyanka Singh ; Arundhuti Sen ; Kevin Francis
Journal of the American Chemical Society 2014 Volume 136(Issue 6) pp:2575-2582
Publication Date(Web):January 22, 2014
DOI:10.1021/ja411998h
Enzyme catalysis has been studied extensively, but the role of enzyme dynamics in the catalyzed chemical conversion is still an enigma. The enzyme dihydrofolate reductase (DHFR) is often used as a model system to assess a network of coupled motions across the protein that may affect the catalyzed chemical transformation. Molecular dynamics simulations, quantum mechanical/molecular mechanical studies, and bioinformatics studies have suggested the presence of a “global dynamic network” of residues in DHFR. Earlier studies of two DHFR distal mutants, G121V and M42W, indicated that these residues affect the chemical step synergistically. While this finding was in accordance with the concept of a network of functional motions across the protein, two residues do not constitute a network. To better define the extent and limits of the proposed network, the current work studied two remote residues predicted to be part of the same network: W133 and F125. The effect of mutations in these residues on the nature of the chemical step was examined via measurements of the temperature-dependence of the intrinsic kinetic isotope effects (KIEs) and other kinetic parameters, and double mutants were used to tie the findings to G121 and M42. The findings indicate that residue F125, which was implicated by both calculations and bioinformatic methods, is a part of the same global dynamic network as G121 and M42, while W133, implicated only by bioinformatics, is not. These findings extend our understanding of the proposed network and the relations between functional and genomic couplings. Delineating that network illuminates the need to consider remote residues and protein structural dynamics in the rational design of drugs and of biomimetic catalysts.
Co-reporter:Zhen Wang ; Priyanka Singh ; Clarissa M. Czekster ; Amnon Kohen ;Vern L. Schramm
Journal of the American Chemical Society 2014 Volume 136(Issue 23) pp:8333-8341
Publication Date(Web):May 12, 2014
DOI:10.1021/ja501936d
The role of fast protein dynamics in enzyme catalysis has been of great interest in the past decade. Recent “heavy enzyme” studies demonstrate that protein mass-modulated vibrations are linked to the energy barrier for the chemical step of catalyzed reactions. However, the role of fast dynamics in the overall catalytic mechanism of an enzyme has not been addressed. Protein mass-modulated effects in the catalytic mechanism of Escherichia coli dihydrofolate reductase (ecDHFR) are explored by isotopic substitution (13C, 15N, and non-exchangeable 2H) of the wild-type ecDHFR (l-DHFR) to generate a vibrationally perturbed “heavy ecDHFR” (h-DHFR). Steady-state, pre-steady-state, and ligand binding kinetics, intrinsic kinetic isotope effects (KIEint) on the chemical step, and thermal unfolding experiments of both l- and h-DHFR show that the altered protein mass affects the conformational ensembles and protein–ligand interactions, but does not affect the hydride transfer at physiological temperatures (25–45 °C). Below 25 °C, h-DHFR shows altered transition state (TS) structure and increased barrier-crossing probability of the chemical step compared with l-DHFR, indicating temperature-dependent protein vibrational coupling to the chemical step. Protein mass-modulated vibrations in ecDHFR are involved in TS interactions at cold temperatures and are linked to dynamic motions involved in ligand binding at physiological temperatures. Thus, mass effects can affect enzymatic catalysis beyond alterations in promoting vibrations linked to chemistry.
Co-reporter:Zahidul Islam ; Timothy S. Strutzenberg ; Ilya Gurevic
Journal of the American Chemical Society 2014 Volume 136(Issue 28) pp:9850-9853
Publication Date(Web):June 20, 2014
DOI:10.1021/ja504341g
Thymidylate synthase (TSase) catalyzes the intracellular de novo formation of thymidylate (a DNA building block) in most living organisms, making it a common target for chemotherapeutic and antibiotic drugs. Two mechanisms have been proposed for the rate-limiting hydride transfer step in TSase catalysis: a stepwise mechanism in which the hydride transfer precedes the cleavage of the covalent bond between the enzymatic cysteine and the product and a mechanism where both happen concertedly. Striking similarities between the enzyme-bound enolate intermediates formed in the initial and final step of the reaction supported the first mechanism, while QM/MM calculations favored the concerted mechanism. Here, we experimentally test these two possibilities using secondary kinetic isotope effect (KIE), mutagenesis study, and primary KIEs. The findings support the concerted mechanism and demonstrate the critical role of an active site arginine in substrate binding, activation of enzymatic nucleophile, and the hydride transfer studied here. The elucidation of this reduction/substitution sheds light on the critical catalytic step in TSase and may aid future drug or biomimetic catalyst design.
Co-reporter:C. Tony Liu;Kevin Francis;Joshua P. Layfield;Xinyi Huang;Sharon Hammes-Schiffer;Stephen J. Benkovic
PNAS 2014 Volume 111 (Issue 51 ) pp:18231-18236
Publication Date(Web):2014-12-23
DOI:10.1073/pnas.1415940111
The reaction catalyzed by Escherichia coli dihydrofolate reductase (ecDHFR) has become a model for understanding enzyme catalysis, and yet several details of its mechanism are still unresolved.
Specifically, the mechanism of the chemical step, the hydride transfer reaction, is not fully resolved. We found, unexpectedly,
the presence of two reactive ternary complexes [enzyme:NADPH:7,8-dihydrofolate (E:NADPH:DHF)] separated by one ionization
event. Furthermore, multiple kinetic isotope effect (KIE) studies revealed a stepwise mechanism in which protonation of the
DHF precedes the hydride transfer from the nicotinamide cofactor (NADPH) for both reactive ternary complexes of the WT enzyme.
This mechanism was supported by the pH- and temperature-independent intrinsic KIEs for the C-H→C hydride transfer between
NADPH and the preprotonated DHF. Moreover, we showed that active site residues D27 and Y100 play a synergistic role in facilitating
both the proton transfer and subsequent hydride transfer steps. Although D27 appears to have a greater effect on the overall
rate of conversion of DHF to tetrahydrofolate, Y100 plays an important electrostatic role in modulating the pKa of the N5 of DHF to enable the preprotonation of DHF by an active site water molecule.
Co-reporter:Daniel Roston
Journal of Labelled Compounds and Radiopharmaceuticals 2014 Volume 57( Issue 2) pp:75-77
Publication Date(Web):
DOI:10.1002/jlcr.3143
Isotopically labeled enzymatic substrates and biological metabolites are useful for many mechanistic analyses, particularly the study of kinetic and equilibrium isotope effects, determining the stereospecificity of enzymes, and resolving metabolic pathways. Here, we present the one-pot synthesis, purification, and kinetic analysis of 7R-[2H]-phenyl-[14C]-benzyl alcohol. The procedure involves a chemoenzymatic synthesis that couples formate dehydrogenase to alcohol dehydrogenase with a catalytic amount of nicotinamide cofactor. The reaction goes to completion overnight, and the measurement of a competitive kinetic isotope effect on the enzymatic oxidation of the purified product identified no 1H contamination. This measurement is very sensitive to such isotopic contamination and verified the high level of isotopic and enantiomeric purity yielded by the new synthetic procedure. Copyright © 2013 John Wiley & Sons, Ltd.
Co-reporter:Daniel Roston
Journal of the American Chemical Society 2013 Volume 135(Issue 37) pp:13624-13627
Publication Date(Web):September 5, 2013
DOI:10.1021/ja405917m
The physical mechanism of C–H bond activation by enzymes is the subject of intense study, and we have tested the predictions of two competing models for C–H activation in the context of alcohol dehydrogenase. The kinetic isotope effects (KIEs) in this enzyme have previously suggested a model of quantum mechanical tunneling and coupled motion of primary (1°) and secondary (2°) hydrogens. Here we measure the 2° H/T KIEs with both H and D at the 1° position and find that the 2° KIE is significantly deflated with D-transfer, consistent with the predictions of recent Marcus-like models of H-transfer. The results suggest that the fast dynamics of H-tunneling result in a 1° isotope effect on the structure of the tunneling ready state: the trajectory of D-transfer goes through a shorter donor–acceptor distance than that of H-transfer.
Co-reporter:Zhen Wang, Silvia Ferrer, Vicent Moliner, and Amnon Kohen
Biochemistry 2013 Volume 52(Issue 13) pp:
Publication Date(Web):March 7, 2013
DOI:10.1021/bi400267q
The cleavage of covalent C–H bonds is one of the most energetically demanding, yet biologically essential, chemical transformations. Two C–H bond cleavages are involved in the reaction catalyzed by thymidylate synthase (TSase), which provides the sole de novo source of thymidylate (i.e., the DNA base T) for most organisms. Our QM/MM free energy calculations show that the C–H → O proton transfer has three transition states that are energetically similar but structurally diverse. These characteristics are different from our previous calculation results on the C–H → C hydride transfer, providing an explanation for differences in temperature dependences of KIEs on these two C–H bond activation steps. The calculations also suggest that the traditionally proposed covalent bond between the protein and substrate (the C6–S bond) is very labile during the multistep catalytic reaction. Collective protein motions not only assist cleavage of the C6–S bond to stabilize the transition state of the proton transfer step but also rearrange the H-bond network at the end of this step to prepare the active site for subsequent chemical steps. These computational results illustrate functionalities of specific protein residues that reconcile many previous experimental observations and provide guidance for future experiments to examine the proposed mechanisms. The synchronized conformational changes in the protein and ligands observed in our simulations demonstrate participation of protein motions in the reaction coordinate of enzymatic reactions. Our computational findings suggest the existence of new reaction intermediates not covalently bound to TSase, which may lead to a new class of drugs targeting DNA biosynthesis.
Co-reporter:Tatiana V. Mishanina ; Eric M. Koehn ; John A. Conrad ; Bruce A. Palfey ; Scott A. Lesley
Journal of the American Chemical Society 2012 Volume 134(Issue 9) pp:4442-4448
Publication Date(Web):January 30, 2012
DOI:10.1021/ja2120822
Thymidylate is a DNA nucleotide that is essential to all organisms and is synthesized by the enzyme thymidylate synthase (TSase). Several human pathogens rely on an alternative flavin-dependent thymidylate synthase (FDTS), which differs from the human TSase both in structure and molecular mechanism. It has recently been shown that FDTS catalysis does not rely on an enzymatic nucleophile and that the proposed reaction intermediates are not covalently bound to the enzyme during catalysis, an important distinction from the human TSase. Here we report the chemical trapping, isolation, and identification of a derivative of such an intermediate in the FDTS-catalyzed reaction. The chemically modified reaction intermediate is consistent with currently proposed FDTS mechanisms that do not involve an enzymatic nucleophile, and it has never been observed during any other TSase reaction. These findings establish the timing of the methylene transfer during FDTS catalysis. The presented methodology provides an important experimental tool for further studies of FDTS, which may assist efforts directed toward the rational design of inhibitors as leads for future antibiotics.
Co-reporter:Zhen Wang ; Thelma Abeysinghe ; Janet S. Finer-Moore ; Robert M. Stroud
Journal of the American Chemical Society 2012 Volume 134(Issue 42) pp:17722-17730
Publication Date(Web):October 3, 2012
DOI:10.1021/ja307859m
The role of protein flexibility in enzyme-catalyzed activation of chemical bonds is an evolving perspective in enzymology. Here we examine the role of protein motions in the hydride transfer reaction catalyzed by thymidylate synthase (TSase). Being remote from the chemical reaction site, the Y209W mutation of Escherichia coli TSase significantly reduces the protein activity, despite the remarkable similarity between the crystal structures of the wild-type and mutant enzymes with ligands representing their Michaelis complexes. The most conspicuous difference between these two crystal structures is in the anisotropic B-factors, which indicate disruption of the correlated atomic vibrations of protein residues in the mutant. This dynamically altered mutant allows a variety of small thiols to compete for the reaction intermediate that precedes the hydride transfer, indicating disruption of motions that preorganize the protein environment for this chemical step. Although the mutation causes higher enthalpy of activation of the hydride transfer, it only shows a small effect on the temperature dependence of the intrinsic KIE, suggesting marginal changes in the geometry and dynamics of the H-donor and -acceptor at the tunneling ready state. These observations suggest that the mutation disrupts the concerted motions that bring the H-donor and -acceptor together during the pre- and re-organization of the protein environment. The integrated structural and kinetic data allow us to probe the impact of protein motions on different time scales of the hydride transfer reaction within a complex enzymatic mechanism.
Co-reporter:Daniel Roston, Christopher M. Cheatum, and Amnon Kohen
Biochemistry 2012 Volume 51(Issue 34) pp:
Publication Date(Web):August 3, 2012
DOI:10.1021/bi300613e
Kinetic isotope effects (KIEs) and their temperature dependence can probe the structural and dynamic nature of enzyme-catalyzed proton or hydride transfers. The molecular interpretation of their temperature dependence requires expensive and specialized quantum mechanics/molecular mechanics (QM/MM) calculations to provide a quantitative molecular understanding. Currently available phenomenological models use a nonadiabatic assumption that is not appropriate for most hydride and proton-transfer reactions, while others require more parameters than the experimental data justify. Here we propose a phenomenological interpretation of KIEs based on a simple method to quantitatively link the size and temperature dependence of KIEs to a conformational distribution of the catalyzed reaction. This model assumes adiabatic hydrogen tunneling, and by fitting experimental KIE data, the model yields a population distribution for fluctuations of the distance between donor and acceptor atoms. Fits to data from a variety of proton and hydride transfers catalyzed by enzymes and their mutants, as well as nonenzymatic reactions, reveal that steeply temperature-dependent KIEs indicate the presence of at least two distinct conformational populations, each with different kinetic behaviors. We present the results of these calculations for several published cases and discuss how the predictions of the calculations might be experimentally tested. This analysis does not replace molecular QM/MM investigations, but it provides a fast and accessible way to quantitatively interpret KIEs in the context of a Marcus-like model.
Co-reporter:Tatiana V. Mishanina, Eric M. Koehn, Amnon Kohen
Bioorganic Chemistry 2012 Volume 43() pp:37-43
Publication Date(Web):August 2012
DOI:10.1016/j.bioorg.2011.11.005
Uracil methylation is essential for survival of organisms and passage of information from generation to generation with high fidelity. Two alternative uridyl methylation enzymes, flavin-dependent thymidylate synthase and folate/FAD-dependent RNA methyltransferase, have joined the long-known classical enzymes, thymidylate synthase and SAM-dependent RNA methyltransferase. These alternative enzymes differ significantly from their classical counterparts in structure, cofactor requirements and chemical mechanism. This review covers the available structural and mechanistic knowledge of the classical and alternative enzymes in biological uracil methylation, and offers a possibility of using inhibitors specifically aiming at microbial thymidylate production as antimicrobial drugs.Graphical abstractHighlights► Uridyl methylation enzymes: thymidylate synthase and RNA methyltransferase. ► Alternative, flavin dependent thymidylate synthase and its mechanism. ► Alternative RNA methyltransferase, its mechanism and biological function. ► Uridyl methylation enzymes as potential antibiotic targets.
Co-reporter:Arundhuti Sen, Vanja Stojković, Amnon Kohen
Analytical Biochemistry 2012 430(2) pp: 123-129
Publication Date(Web):15 November 2012
DOI:10.1016/j.ab.2012.08.012
14C-labeled nicotinamide cofactors are widely employed in biomedical investigations, for example, to delineate metabolic pathways, to elucidate enzymatic mechanisms, and as substrates in kinetic isotope effect (KIE) experiments. The 14C label has generally been located remote from the reactive position, frequently at the adenine ring. Rising costs of commercial precursors and disruptions in the availability of enzymes required for established syntheses have recently made the preparation of labeled nicotinamides such as [Ad-14C]NADPH unviable. Here, we report the syntheses and characterization of several alternatives: [carbonyl-14C]NADPH, 4R-[carbonyl-14C, 4-2H]NADPH, and [carbonyl-14C, 4-2H2]NADPH. The new procedures use [carbonyl-14C]nicotinamide as starting material, because it is significantly cheaper than other commercial 14C precursors of NADPH, and require only one commercially available enzyme to prepare NAD(P)+ and NAD(P)H. The proximity of carbonyl-14C to the reactive center raises the risk of an inopportune 14C isotope effect. This concern has been alleviated via competitive KIE measurements with Escherichia coli dihydrofolate reductase (EcDHFR) that use this specific carbonyl-14C NADPH. A combination of binding isotope effect and KIE measurements yielded no significant 12C/14C isotope effect at the amide carbonyl (KIE = 1.003 ± 0.004). The reported procedure provides a high-yield, high-purity, and cost-effective alternative to labeled nicotinamide cofactors synthesized by previously published routes.
Co-reporter:Qi Liu, Yu Zhao, Blake Hammann, James Eilers, Yun Lu, and Amnon Kohen
The Journal of Organic Chemistry 2012 Volume 77(Issue 16) pp:6825-6833
Publication Date(Web):July 26, 2012
DOI:10.1021/jo300879r
To assess the contribution of physical features to enzyme catalysis, the enzymatic reaction has to be compared to a relevant uncatalyzed reaction. While such comparisons have been conducted for some hydrolytic and radical reactions, it is most challenging for biological hydride transfer and redox reactions in general. Here, the same experimental tools used to study the H-tunneling and coupled motions for enzymatic hydride transfer between two carbons were used in the study of an uncatalyzed model reaction. The enzymatic oxidations of benzyl alcohol and its substituted analogues mediated by alcohol dehydrogenases were compared to the oxidations by 9-phenylxanthylium cation (PhXn+). The PhXn+serves as an NAD+ model, while the solvent, acetonitrile, models the protein environment. Experimental comparisons included linear free energy relations with Hammett reaction constant (ρ) of zero versus −2.7; temperature-independent versus temperature-dependent primary KIEs; deflated secondary KIEs with deuteride transfer (i.e., primary-secondary coupled motion) versus no coupling between secondary KIEs and H- or D-transfer; and large versus small secondary KIEs for the enzymatic versus uncatalyzed alcohol oxidation. Some of the differences may come from differences in the order of microscopic steps between the catalyzed versus uncatalyzed reactions. However, several of these comparative experiments indicate that in contrast to the uncatalyzed reaction the transition state of the enzymatic reaction is better reorganized for H-tunneling and its H-donor is better rehybridized prior to the C–H→C transfer. These findings suggest an important role for these physical features in enzyme catalysis.
Co-reporter:Eric M. Koehn;Arjun Prabhakar;Scott A. Lesley;Laura L. Perissinotti;Irimpan I. Mathews;Salah Moghram
PNAS 2012 Volume 109 (Issue 39 ) pp:15722-15727
Publication Date(Web):2012-09-25
DOI:10.1073/pnas.1206077109
The DNA nucleotide thymidylate is synthesized by the enzyme thymidylate synthase, which catalyzes the reductive methylation
of deoxyuridylate using the cofactor methylene-tetrahydrofolate (CH2H4folate). Most organisms, including humans, rely on the thyA- or TYMS-encoded classic thymidylate synthase, whereas, certain microorganisms, including all Rickettsia and other pathogens, use an alternative thyX-encoded flavin-dependent thymidylate synthase (FDTS). Although several crystal structures of FDTSs have been reported, the
absence of a structure with folates limits understanding of the molecular mechanism and the scope of drug design for these
enzymes. Here we present X-ray crystal structures of FDTS with several folate derivatives, which together with mutagenesis,
kinetic analysis, and computer modeling shed light on the cofactor binding and function. The unique structural data will likely
facilitate further elucidation of FDTSs’ mechanism and the design of structure-based inhibitors as potential leads to new
antimicrobial drugs.
Co-reporter:Lindsay R. Ditzler ; Arundhuti Sen ; Michael J. Gannon ; Amnon Kohen ;Alexei V. Tivanski
Journal of the American Chemical Society 2011 Volume 133(Issue 34) pp:13284-13287
Publication Date(Web):August 2, 2011
DOI:10.1021/ja205409v
Escherichia coli dihydrofolate reductase (ecDHFR) has one surface cysteine, C152, located opposite and distal to the active site. Here, we show that the enzyme spontaneously assembles on an ultraflat gold surface as a homogeneous, covalently bound monolayer. Surprisingly, the activity of the gold-immobilized ecDHFR as measured by radiographic analysis was found to be similar to that of the free enzyme in solution. Molecular recognition force spectroscopy was used to study the dissociation forces involved in the rupture of AFM probe-tethered methotrexate (MTX, a tight-binding inhibitor of DHFR) from the gold-immobilized enzyme. Treatment of the ecDHFR monolayer with free MTX diminished the interaction of the functionalized tip with the surface, suggesting that the interaction was indeed active-site specific. These findings demonstrate the viability of a simple and direct enzymatic surface-functionalization without the use of spacers, thus, opening the door to further applications in the area of biomacromolecular force spectroscopy.
Co-reporter:Vanja Stojković ; Laura L. Perissinotti ; Daniel Willmer ; Stephen J. Benkovic
Journal of the American Chemical Society 2011 Volume 134(Issue 3) pp:1738-1745
Publication Date(Web):December 15, 2011
DOI:10.1021/ja209425w
A significant contemporary question in enzymology involves the role of protein dynamics and hydrogen tunneling in enhancing enzyme catalyzed reactions. Here, we report a correlation between the donor–acceptor distance (DAD) distribution and intrinsic kinetic isotope effects (KIEs) for the dihydrofolate reductase (DHFR) catalyzed reaction. This study compares the nature of the hydride-transfer step for a series of active-site mutants, where the size of a side chain that modulates the DAD (I14 in E. coli DHFR) is systematically reduced (I14V, I14A, and I14G). The contributions of the DAD and its dynamics to the hydride-transfer step were examined by the temperature dependence of intrinsic KIEs, hydride-transfer rates, activation parameters, and classical molecular dynamics (MD) simulations. Results are interpreted within the framework of the Marcus-like model where the increase in the temperature dependence of KIEs arises as a direct consequence of the deviation of the DAD from its distribution in the wild type enzyme. Classical MD simulations suggest new populations with larger average DADs, as well as broader distributions, and a reduction in the population of the reactive conformers correlated with the decrease in the size of the hydrophobic residue. The more flexible active site in the mutants required more substantial thermally activated motions for effective H-tunneling, consistent with the hypothesis that the role of the hydrophobic side chain of I14 is to restrict the distribution and dynamics of the DAD and thus assist the hydride-transfer. These studies establish relationships between the distribution of DADs, the hydride-transfer rates, and the DAD’s rearrangement toward tunneling-ready states. This structure–function correlation shall assist in the interpretation of the temperature dependence of KIEs caused by mutants far from the active site in this and other enzymes, and may apply generally to C–H→C transfer reactions.
Co-reporter:Arundhuti Sen, Atsushi Yahashiri, and Amnon Kohen
Biochemistry 2011 Volume 50(Issue 29) pp:
Publication Date(Web):June 20, 2011
DOI:10.1021/bi2003873
Kinetic isotope effect (KIE) studies can provide insight into the mechanism and kinetics of specific chemical steps in complex catalytic cascades. Recent results from hydrogen KIE measurements have examined correlations between enzyme dynamics and catalytic function, leading to a surge of studies in this area. Unfortunately, most enzymatic H-transfer reactions are not rate limiting, and the observed KIEs do not reliably reflect the intrinsic KIEs on the chemical step of interest. Given their importance to understanding the chemical step under study, accurate determination of the intrinsic KIE from the observed data is essential. In 1975, Northrop developed an elegant method to assess intrinsic KIEs from their observed values [Northrop, D. B. (1975) Steady-state analysis of kinetic isotope effects in enzymic reactions. Biochemistry14, 2644−2651]. The Northrop method involves KIE measurements using all three hydrogen isotopes, where one of them serves as the reference isotope. This method has been successfully used with different combinations of observed KIEs over the years, but criteria for a rational choice of reference isotope have never before been experimentally determined. Here we compare different reference isotopes (and hence distinct experimental designs) using the reduction of dihydrofolate and dihydrobiopterin by two dissimilar enzymes as model reactions. A number of isotopic labeling patterns have been applied to facilitate the comparative study of reference isotopes. The results demonstrate the versatility of the Northrop method and that such experiments are limited only by synthetic techniques, availability of starting materials, and the experimental error associated with the use of distinct combinations of isotopologues.
Co-reporter:Zhen Wang
Journal of the American Chemical Society 2010 Volume 132(Issue 28) pp:9820-9825
Publication Date(Web):June 24, 2010
DOI:10.1021/ja103010b
Examination of the nature of different bond activations along the same catalytic path is of general interest in chemistry and biology. In this report, we compare the physical nature of two sequential H-transfers in the same enzymatic reaction. Thymidylate synthase (TSase) catalyzes a complex reaction that involves many chemical transformations including two different C−H bond cleavages, a rate-limiting C−H−C hydride transfer and a non-rate-limiting C−H−O proton transfer. Although the large kinetic complexity imposes difficulties in studying the proton transfer catalyzed by TSase, we are able to experimentally extract the intrinsic kinetic isotope effects (KIEs) on both steps. In contrast with the hydride transfer, the intrinsic KIEs of the proton transfer are temperature dependent. The results are interpreted within the framework of the Marcus-like model. This interpretation suggests that TSase optimizes the donor−acceptor geometries for the slower and overall rate-limiting hydride transfer but not for the faster proton transfer.
Co-reporter:Vanja Stojković, Laura L. Perissinotti, Jeeyeon Lee, Stephen J. Benkovic and Amnon Kohen
Chemical Communications 2010 vol. 46(Issue 47) pp:8974-8976
Publication Date(Web):25 Oct 2010
DOI:10.1039/C0CC02988B
Comparison of the nature of hydride transfer in wild-type and active site mutant (I14A) of dihydrofolate reductase suggests that the size of this side chain at position 14 modulates H-tunneling.
Co-reporter:Samrat Dutta, Richard J. Cook, Jon C.D. Houtman, Amnon Kohen, Christopher M. Cheatum
Analytical Biochemistry 2010 Volume 407(Issue 2) pp:241-246
Publication Date(Web):15 December 2010
DOI:10.1016/j.ab.2010.08.008
Enzyme active-site dynamics at femtosecond to picosecond time scales are of great biochemical importance, but remain relatively unexplored due to the lack of appropriate analytical methods. Two-dimensional infrared (2D IR) spectroscopy is one of the few methods that can examine chemical biological motions at this time scale, but all the IR probes used so far were specific to a few unique enzymes. The lack of IR probes of broader specificity is a major limitation to further 2D IR studies of enzyme dynamics. Here we describe the synthesis of a general IR probe for nicotinamide-dependent enzymes. This azido analog of the ubiquitous cofactor nicotinamide adenine dinucleotide is found to be stable and bind to several dehydrogenases with dissociation constants similar to that for the native cofactor. The infrared absorption spectra of this probe bound to several enzymes indicate that it has significant potential as a 2D IR probe to investigate femtosecond dynamics of nicotinamide-dependent enzymes.
Co-reporter:Arundhuti Sen
Journal of Physical Organic Chemistry 2010 Volume 23( Issue 7) pp:613-619
Publication Date(Web):
DOI:10.1002/poc.1633
Abstract
Kinetic isotope effects (KIEs), originally a tool for the physical organic chemist and mechanistic enzymologist, have been instrumental in furthering our understanding of quantum-mechanical hydrogen tunneling in enzymatic systems. This review focuses on the use of KIE studies to investigate this phenomenon in enzyme-catalyzed reactions. A number of enzymes wherein KIEs have been used as a probe for H-tunneling are discussed, including dihydrofolate reductase (DHFR), alcohol dehydrogenase (ADH), and formate dehydrogenase (FDH). Particular emphasis has been placed on the significance of KIE results in exposing the chemical H-transfer step in the complex kinetic cascades typical to these systems, as well as on questions regarding the influence of protein dynamics on tunneling and, consequently, on the whole enzymatic reaction. Copyright © 2009 John Wiley & Sons, Ltd.
Co-reporter:Daniel Roston
PNAS 2010 107 (21 ) pp:9572-9577
Publication Date(Web):2010-05-25
DOI:10.1073/pnas.1000931107
For several decades the hydride transfer catalyzed by alcohol dehydrogenase has been difficult to understand. Here we add
to the large corpus of anomalous and paradoxical data collected for this reaction by measuring a normal (> 1) 2° kinetic isotope
effect (KIE) for the reduction of benzaldehyde. Because the relevant equilibrium effect is inverse (< 1), this KIE eludes
the traditional interpretation of 2° KIEs. It does, however, enable the development of a comprehensive model for the “tunneling
ready state” (TRS) of the reaction that fits into the general scheme of Marcus-like models of hydrogen tunneling. The TRS
is the ensemble of states along the intricate reorganization coordinate, where H tunneling between the donor and acceptor
occurs (the crossing point in Marcus theory). It is comparable to the effective transition state implied by ensemble-averaged
variational transition state theory. Properties of the TRS are approximated as an average of the individual properties of
the donor and acceptor states. The model is consistent with experimental findings that previously appeared contradictory;
specifically, it resolves the long-standing ambiguity regarding the location of the TRS (aldehyde-like vs. alcohol-like).
The new picture of the TRS for this reaction identifies the principal components of the collective reaction coordinate and
the average structure of the saddle point along that coordinate.
Co-reporter:Daniel Roston
PNAS 2010 107 (21 ) pp:9572-9577
Publication Date(Web):2010-05-25
DOI:10.1073/pnas.1000931107
For several decades the hydride transfer catalyzed by alcohol dehydrogenase has been difficult to understand. Here we add
to the large corpus of anomalous and paradoxical data collected for this reaction by measuring a normal (> 1) 2° kinetic isotope
effect (KIE) for the reduction of benzaldehyde. Because the relevant equilibrium effect is inverse (< 1), this KIE eludes
the traditional interpretation of 2° KIEs. It does, however, enable the development of a comprehensive model for the “tunneling
ready state” (TRS) of the reaction that fits into the general scheme of Marcus-like models of hydrogen tunneling. The TRS
is the ensemble of states along the intricate reorganization coordinate, where H tunneling between the donor and acceptor
occurs (the crossing point in Marcus theory). It is comparable to the effective transition state implied by ensemble-averaged
variational transition state theory. Properties of the TRS are approximated as an average of the individual properties of
the donor and acceptor states. The model is consistent with experimental findings that previously appeared contradictory;
specifically, it resolves the long-standing ambiguity regarding the location of the TRS (aldehyde-like vs. alcohol-like).
The new picture of the TRS for this reaction identifies the principal components of the collective reaction coordinate and
the average structure of the saddle point along that coordinate.
Co-reporter:Vanja Stojkovi&x107;
Israel Journal of Chemistry 2009 Volume 49( Issue 2) pp:163-173
Publication Date(Web):
DOI:10.1560/IJC.49.2.163
Abstract
Many hydrogen transfer processes exhibit nonclassical behavior due to inherent quantum mechanical properties of the hydrogen. Investigation of various enzymes under physiological conditions indicates that hydrogen transfer processes often show significant quantum mechanical behavior. Traditionally, this phenomenon was treated in terms of a tunneling correction to classical or semiclassical models. However, more recently, it has been observed that increasing numbers of enzymes yield data that cannot be rationalized by tunneling correction models. Observations such as large kinetic isotope effects (KIEs) with unusual temperature dependence, isotope effects on Arrhenius preexponential factors with values different from semiclassical predicted ranges, and small temperature-independent KIEs for processes with significant energy of activation, could only be explained with full tunneling models for H transfer. Full tunneling models presume that the solvent or protein fluctuations generate a reactive configuration along the heavy-atom coordinate, from which the hydrogen is transferred through quantum mechanical tunneling. These models are sometimes denoted as environmentally coupled tunneling (ECT) or Marcus-like models, and they link protein dynamics to the catalyzed H transfer. Several enzymatic systems (dihydrofolate reductase, thymidylate synthase, and soybean lipoxygenase) are presented as case studies of proton, hydrogen, and hydride transfer.
Co-reporter:Atsushi Yahashiri;Guy Nimrod Dr.;Nir Ben-Tal ;Elizabeth E. Howell
ChemBioChem 2009 Volume 10( Issue 16) pp:2620-2623
Publication Date(Web):
DOI:10.1002/cbic.200900451
Co-reporter:Atsushi Yahashiri;Arundhuti Sen
Journal of Labelled Compounds and Radiopharmaceuticals 2009 Volume 52( Issue 11) pp:463-466
Publication Date(Web):
DOI:10.1002/jlcr.1660
Abstract
We present a one-pot chemo-enzymatic microscale synthesis of NADPH with two different patterns of isotopic labels: (4R)-[Ad-14C,4-2H] NADPH and (4R)-[Ad-3H,4-2H] NADPH. These co-factors are required by an enormous range of enzymes, and isotopically labeled nicotinamides are consequently of significant interest to researchers. In the current procedure, [Ad-14C] NAD+ and [Ad-3H] NAD+ were phosphorylated by NAD+ kinase to produce [Ad-14C] NADP+ and [Ad-3H] NADP+, respectively. Thermoanaerobium brockii alcohol dehydrogenase was then used to stereospecifically transfer deuterium from C2 of isopropanol to the re face of C4 of NADP+. After purification by HPLC, NMR analysis indicated that the deuterium content at the 4R position is more than 99.7%. The labeled cofactors were then used to successfully and sensitively measure kinetic isotope effects for R67 dihydrofolate reductase, providing strong evidence for the utility of this synthetic methodology. Copyright © 2009 John Wiley & Sons, Ltd.
Co-reporter:Eric M. Koehn,
Todd Fleischmann,
John A. Conrad,
Bruce A. Palfey,
Scott A. Lesley,
Irimpan I. Mathews
&
Amnon Kohen
Nature 2009 458(7240) pp:919
Publication Date(Web):2009-04-16
DOI:10.1038/nature07973
This paper reports an example of thymidylate biosynthesis that occurs without an enzymatic nucleophile, and is found in organisms containing the thyX gene (encoding a flavin-dependent thymidylate synthase). Because several human pathogens depend on this biosynthetic pathway for DNA biosynthesis, it may be possible to develop new, highly selective antibiotics that target this enzyme.
Co-reporter:Atsushi Yahashiri;Elizabeth E. Howell
ChemPhysChem 2008 Volume 9( Issue 7) pp:980-982
Publication Date(Web):
DOI:10.1002/cphc.200800067
Co-reporter:Anatoly Chernyshev, Todd Fleischmann, Eric M. Koehn, Scott A. Lesley and Amnon Kohen
Chemical Communications 2007 (Issue 27) pp:2861-2863
Publication Date(Web):25 Apr 2007
DOI:10.1039/B700977A
New findings lead to a revised understanding of the substrates' binding order, the role of the substrate as an activator, and the observed lag phase in the FDTS catalyzed reaction.
Co-reporter:Anatoly Chernyshev;Todd Fleischmann
Applied Microbiology and Biotechnology 2007 Volume 74( Issue 2) pp:282-289
Publication Date(Web):2007 February
DOI:10.1007/s00253-006-0763-1
The two long-known “classical” enzymes of uridyl-5-methylation, thymidylate synthase and ribothymidyl synthase, have been joined by two alternative methylation enzymes, flavin-dependent thymidylate synthase and folate-dependent ribothymidyl synthase. These two newly discovered enzymes have much in common: both contain flavin cofactors, utilize methylenetetrahydrofolate as a source of methyl group, and perform thymidylate synthesis via chemical pathways distinct from those of their classic counterparts. Several severe human pathogens (e.g., typhus, anthrax, tuberculosis, and more) depend on these “alternative” enzymes for reproduction. These and other distinctive properties make the alternative enzymes and their corresponding genes appealing targets for new antibiotics.
Co-reporter:Aaron Mason, Nitish Agrawal, M. Todd Washington, Scott A. Lesley and Amnon Kohen
Chemical Communications 2006 (Issue 16) pp:1781-1783
Publication Date(Web):17 Mar 2006
DOI:10.1039/B517881A
An unexpected substrate-dependent lag-phase, found in the single turnover reduction of FDTS bound flavin, sheds light on the molecular mechanism of this alternative thymidylate synthase.
Co-reporter:Lin Wang;Nina M. Goodey;Stephen J. Benkovic
PNAS 2006 Volume 103 (Issue 43 ) pp:15753-15758
Publication Date(Web):2006-10-24
DOI:10.1073/pnas.0606976103
One of the most intriguing questions in modern enzymology is whether enzyme dynamics evolved to enhance the catalyzed chemical
transformation. In this study, dihydrofolate reductase, a small monomeric protein that catalyzes a single C-H–C transfer,
is used as a model system to address this question. Experimental and computational studies have proposed a dynamic network
that includes two residues remote from the active site (G121 and M42). The current study compares the nature of the H-transfer
step of the WT enzyme, two single mutants, and their double mutant. The contribution of quantum mechanical tunneling and enzyme
dynamics to the H-transfer step was examined by determining intrinsic kinetic isotope effects, their temperature dependence,
and activation parameters. Different patterns of environmentally coupled tunneling were found for these four enzymes. The
findings indicate that the naturally evolved WT dihydrofolate reductase requires no donor–acceptor distance fluctuations (no
gating). Both single mutations affect the rearrangement of the system before tunneling, so some gating is required, but the
overall nature of the environmentally coupled tunneling appears similar to that of the WT enzyme. The double mutation, on
the other hand, seems to cause a major change in the nature of H transfer, leading to poor reorganization and substantial
gating. These findings support the suggestion that these distal residues synergistically affect the H transfer at the active
site of the enzyme. This observation is in accordance with the notion that these remote residues are part of a dynamic network
that is coupled to the catalyzed chemistry.
Co-reporter:Amnon Kohen;Baoyu Hong
Journal of Labelled Compounds and Radiopharmaceuticals 2005 Volume 48(Issue 10) pp:759-769
Publication Date(Web):25 JUL 2005
DOI:10.1002/jlcr.993
A one-pot chemo-enzymatic microscale synthesis of isotopically labeled R-[6-YH; 11-XH] N5, N10 methylene-5, 6, 7, 8-tetrahydrofolate (CH2H4folate) is presented, where Y=1 or 2 represents protium or deuterium, and X=1, 2 or 3 represents protium, deuterium or tritium, respectively. In this procedure, Thermoanaerobium brockii alcohol dehydrogenase (tbADH) and Escherichia coli dihydrofolate reductase (ecDHFR) were used simultaneously in the reaction mixture. First, tbADH stereospecifically catalyzes a hydride transfer from [2-YH] iPrOH to the re face of C-4 NADP+. The ecDHFR then reduced 7, 8-dihydrofolate (H2folate) to form (6S)-H4folate. Finally, the enzymatic reactions were followed by chemical trapping with isotopically labeled formaldehyde ([XH]-HCHO) to form the final product. The preparation of deuterium- and tritium-labeled formaldehyde is also presented. Two reverse phase HPLC methods were developed for analysis and purification of product R-[6-YH; 11-XH] CH2H4folate. This isotopically labeled cofactor can be used to study 1° and 2° kinetic isotope effects (KIEs) with any CH2H4folate dependent enzyme as demonstrated by studies with E. coli thymidylate synthase (TS). Copyright © 2005 John Wiley & Sons, Ltd.
Co-reporter:Donald G. Truhlar
PNAS 2001 Volume 98 (Issue 3 ) pp:848-851
Publication Date(Web):2001-01-30
DOI:10.1073/pnas.98.3.848
This paper draws attention to selected experiments on
enzyme-catalyzed reactions that show convex Arrhenius plots, which are
very rare, and points out that Tolman's interpretation of the
activation energy places a fundamental model-independent constraint on
any detailed explanation of these reactions. The analysis presented
here shows that in such systems, the rate coefficient as a function of
energy is not just increasing more slowly than expected, it is actually
decreasing. This interpretation of the data provides a constraint on
proposed microscopic models, i.e., it requires that any successful
model of a reaction with a convex Arrhenius plot should be consistent
with the microcanonical rate coefficient being a decreasing function of
energy. The implications and limitations of this analysis to
interpreting enzyme mechanisms are discussed. This model-independent
conclusion has broad applicability to all fields of kinetics, and we
also draw attention to an analogy with diffusion in metastable fluids
and glasses.
Co-reporter:Priyanka Singh, Holly Morris, Alexei V. Tivanski, Amnon Kohen
Data in Brief (September 2015) Volume 4() pp:19-21
Publication Date(Web):1 September 2015
DOI:10.1016/j.dib.2015.04.005
An assay was developed for measuring the active-site concentration, activity, and thereby the catalytic turnover rate (kcat) of an immobilized dihydrofolate reductase model system (Singh et al., (2015), Anal. Biochem). This data article contains a calibration plot for the developed assay. In the calibration plot rate is plotted as a function of DHFR concentration and shows linear relationship. The concentration of immobilized enzyme was varied by using 5 different size mica chips. The dsDNA concentration was the same for all chips, assuming that the surface area of the mica chip dictates the resulting amount of bound enzyme (i.e. larger sized chip would have more bound DHFR). The activity and concentration of each chip was measured.
Co-reporter:Kevin Francis, Amnon Kohen
Perspectives in Science (May 2014) Volume 1(Issues 1–6) pp:110-120
Publication Date(Web):1 May 2014
DOI:10.1016/j.pisc.2014.02.009
Kinetic isotope effects (KIEs) are a powerful tool in the study of enzymatic mechanism and function. Standardization of how data are reported would allow for comparisons of data collected under different conditions, in different laboratories, or by different assays, and will contribute to the assessment of their fit to different models and support of quantitative and qualitative conclusions. This paper will provide suggestions and examples as to how KIE data should be presented in publications of enzyme reaction mechanisms. The importance of following the Standards for the Reporting of Enzymological Data committee׳s recommendations when reporting KIE studies are outlined and a particular focus is placed on procedures for calculating, propagating, and reporting experimental errors. We hope that this paper will be useful for researchers, authors of scientific papers and presentations, and reviewers of these papers alike, and will eventually enhance the impact of KIE studies by assisting in broader application and implications of data.
Co-reporter:Amnon Kohen
Perspectives in Science (December 2016) Volume 9() pp:60-66
Publication Date(Web):1 December 2016
DOI:10.1016/j.pisc.2016.03.023
Examination of the chemical step catalysed by dihydrofolate reductase (DHFR) suggested preservation of an “ideal” transition state as the enzyme evolves from bacteria to human. This observation is enigmatic: since evolutionary pressure is most effective on enzymes’ second order rate constant (kcat/KM) and since the chemistry is not rate limiting on that kinetic parameter, why is the nature of the chemical step preserved? Studies addressing this question were presented in the 2015 Beilstein ESCEC Symposium and are summarized below.
Co-reporter:Daniel Roston, Zahidul Islam, Amnon Kohen
Archives of Biochemistry and Biophysics (15 February 2014) Volume 544() pp:96-104
Publication Date(Web):15 February 2014
DOI:10.1016/j.abb.2013.10.010
Co-reporter:Zhen Wang ; Paul J. Sapienza ; Thelma Abeysinghe ; Calvin Luzum ; Andrew L. Lee ; Janet S. Finer-Moore ; Robert M. Stroud
Journal of the American Chemical Society () pp:
Publication Date(Web):April 23, 2013
DOI:10.1021/ja400761x
Thymidylate synthase (TSase) produces the sole intracellular de novo source of thymidine (i.e., the DNA base T) and thus is a common target for antibiotic and anticancer drugs. Mg2+ has been reported to affect TSase activity, but the mechanism of this interaction has not been investigated. Here we show that Mg2+ binds to the surface of Escherichia coli TSase and affects the kinetics of hydride transfer at the interior active site (16 Å away). Examination of the crystal structures identifies a Mg2+ near the glutamyl moiety of the folate cofactor, providing the first structural evidence for Mg2+ binding to TSase. The kinetics and NMR relaxation experiments suggest that the weak binding of Mg2+ to the protein surface stabilizes the closed conformation of the ternary enzyme complex and reduces the entropy of activation on the hydride transfer step. Mg2+ accelerates the hydride transfer by ∼7-fold but does not affect the magnitude or temperature dependence of the intrinsic kinetic isotope effect. These results suggest that Mg2+ facilitates the protein motions that bring the hydride donor and acceptor together, but it does not change the tunneling ready state of the hydride transfer. These findings highlight how variations in cellular Mg2+ concentration can modulate enzyme activity through long-range interactions in the protein, rather than binding at the active site. The interaction of Mg2+ with the glutamyl tail of the folate cofactor and nonconserved residues of bacterial TSase may assist in designing antifolates with polyglutamyl substitutes as species-specific antibiotic drugs.
Co-reporter:Tatiana V. Mishanina ; John M. Corcoran
Journal of the American Chemical Society () pp:
Publication Date(Web):July 15, 2014
DOI:10.1021/ja506108b
Thymidylate is a critical DNA nucleotide that has to be synthesized in cells de novo by all organisms. Flavin-dependent thymidylate synthase (FDTS) catalyzes the final step in this de novo production of thymidylate in many human pathogens, but it is absent from humans. The FDTS reaction proceeds via a chemical route that is different from its human enzyme analogue, making FDTS a potential antimicrobial target. The chemical mechanism of FDTS is still not understood, and the two most recently proposed mechanisms involve reaction intermediates that are unusual in pyrimidine biosynthesis and biology in general. These mechanisms differ in the relative timing of the reaction of the flavin with the substrate. The consequence of this difference is significant: the intermediates are cationic in one case and neutral in the other, an important consideration in the construction of mechanism-based enzyme inhibitors. Here we test these mechanisms via chemical trapping of reaction intermediates, stopped-flow, and substrate hydrogen isotope exchange techniques. Our findings suggest that an initial activation of the pyrimidine substrate by reduced flavin is required for catalysis, and a revised mechanism is proposed on the basis of previous and new data. These findings and the newly proposed mechanism add an important piece to the puzzle of the mechanism of FDTS and suggest a new class of intermediates that, in the future, may serve as targets for mechanism-based design of FDTS-specific inhibitors.
Co-reporter:Anatoly Chernyshev, Todd Fleischmann, Eric M. Koehn, Scott A. Lesley and Amnon Kohen
Chemical Communications 2007(Issue 27) pp:NaN2863-2863
Publication Date(Web):2007/04/25
DOI:10.1039/B700977A
New findings lead to a revised understanding of the substrates' binding order, the role of the substrate as an activator, and the observed lag phase in the FDTS catalyzed reaction.
Co-reporter:Vanja Stojković, Laura L. Perissinotti, Jeeyeon Lee, Stephen J. Benkovic and Amnon Kohen
Chemical Communications 2010 - vol. 46(Issue 47) pp:NaN8976-8976
Publication Date(Web):2010/10/25
DOI:10.1039/C0CC02988B
Comparison of the nature of hydride transfer in wild-type and active site mutant (I14A) of dihydrofolate reductase suggests that the size of this side chain at position 14 modulates H-tunneling.
Co-reporter:Ananda K. Ghosh, Zahidul Islam, Jonathan Krueger, Thelma Abeysinghe and Amnon Kohen
Physical Chemistry Chemical Physics 2015 - vol. 17(Issue 46) pp:NaN30875-30875
Publication Date(Web):2015/04/15
DOI:10.1039/C5CP01246E
The enzyme thymidylate synthase (TSase), an important chemotherapeutic drug target, catalyzes the formation of 2′-deoxythymidine-5′-monophosphate (dTMP), a precursor of one of the DNA building blocks. TSase catalyzes a multi-step mechanism that includes the abstraction of a proton from the C5 of the substrate 2′-deoxyuridine-5′-monophosphate (dUMP). Previous studies on ecTSase proposed that an active-site residue, Y94 serves the role of the general base abstracting this proton. However, since Y94 is neither very basic, nor connected to basic residues, nor located close enough to the pyrimidine proton to be abstracted, the actual identity of this base remains enigmatic. Based on crystal structures, an alternative hypothesis is that the nearest potential proton-acceptor of C5 of dUMP is a water molecule that is part of a hydrogen bond (H-bond) network comprised of several water molecules and several protein residues including H147, E58, N177, and Y94. Here, we examine the role of the residue Y94 in the proton abstraction step by removing its hydroxyl group (Y94F mutant). We investigated the effect of the mutation on the temperature dependence of intrinsic kinetic isotope effects (KIEs) and found that these KIEs are more temperature dependent than those of the wild-type enzyme (WT). These results suggest that the phenolic –OH of Y94 is a component of the transition state for the proton abstraction step. The findings further support the hypothesis that no single functional group is the general base, but a network of bases and hydroxyls (from water molecules and tyrosine) sharing H-bonds across the active site can serve the role of the general base to remove the pyrimidine proton.
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