Aldol Cleavage in Water and the Power of Citrate Lyase as a Catalyst.

Citrate lyase allows Klebsiella aerogenes to grow anaerobically on citrate as the sole carbon source. Arrhenius analysis of experiments at high temperatures indicates that citrate is cleaved nonenzymatically to acetate and oxaloacetate with a t1/2 of 6.9 million years in neutral solution at 25 °C, while malate cleavage occurs even more slowly (t1/2 = 280 million years). However, t1/2 is only 10 days for the nonenzymatic cleavage of 4-hydroxy-2-ketoglutarate, indicating that the introduction of an α-keto group enhances the rate of aldol cleavage of malate by a factor of 1010. The aldol cleavages of citrate and malate, like the decarboxylation of malonate (t1/2 = 180 years), are associated with a near-zero entropy of activation, and their extreme differences in rate reflect differences between their heats of activation. Citrate lyase enhances the rate of substrate cleavage 6 × 1015-fold, comparable in magnitude with the rate enhancement produced by OMP decarboxylase, although these enzymes are strikingly different in their mechanisms of action.

The Burden Borne by Protein Methyltransferases: Rates and Equilibria of Non-enzymatic Methylation of Amino Acid Side Chains by SAM in Water.

SAM is a powerful methylating agent, with a methyl group transfer potential matching the phosphoryl group transfer potential of ATP. SAM-dependent N-methyltransferases have evolved to catalyze the modification of specific lysine residues in histones and transcription factors, in addition to generating epinephrine, N-methylnicotinamide, and a quaternary amine (betaine) that is used to maintain osmotic pressure in plants and halophilic bacteria. To assess the catalytic power of these enzymes and their potential susceptibility to transition state and multisubstrate analogue inhibitors, we determined the rates and positions of the equilibrium of methyl transfer from the trimethylsulfonium ion to model amines in the absence of a catalyst. Unlike the methyl group transfer potential of SAM, which becomes more negative with an increase in pH throughout the normal pH range, equilibrium constants for the hydrolytic demethylation of secondary, tertiary, and quaternary amines are found to be insensitive to a change in pH and resemble each other in magnitude, with an average ΔG value of approximately -0.7 kcal/mol at pH 7. Thus, each of the three steps in the mono-, di-, and trimethylation of lysine by SAM is accompanied by a change in free energy of -7.5 kcal/mol in a neutral solution. Arrhenius analysis of the uncatalyzed reactions shows that the unprotonated form of glycine attacks the trimethylsulfonium ion (TMS+) with second-order rates constant of 1.8 × 10-7 M-1 s-1 at 25 °C (ΔH⧧ = 22 kcal/mol, and TΔS⧧ = -6 kcal/mol). Comparable values are observed for the methylation of secondary and tertiary amines, with k25 values of 1.1 × 10-7 M-1 s-1 for sarcosine and 4.3 × 10-8 M-1 s-1 for dimethylglycine. The non-enzymatic methylations of imidazole and methionine by TMS+, benchmarks for the methylation of histidine and methionine residues by SETD3, exhibit k25 values of 3.3 × 10-9 and 1.2 × 10-9 M-1 s-1, respectively. Lysine methylation by SAM, although slow under physiological conditions (t1/2 = 7 weeks at 25 °C), is accelerated 1.1 × 1012 -fold at the active site of a SET domain methyltransferase.

Ether Hydrolysis, Ether Thiolysis, and the Catalytic Power of Etherases in the Disassembly of Lignin.

The recycling of much of the carbon in Nature depends on the breakdown of polymers in woody matter, notably cellulose (a polyacetal) and lignin (a polyether). Here, we show that equilibrium favors ether hydrolysis in water, although the rates of spontaneous hydrolysis of ethers are too slow to measure in neutral solution except at temperatures approaching the critical point of water. Circumventing that kinetic obstacle, glutathione-dependent etherases from white-rot fungi are known to employ the thiolate group of glutathione to attack guaiacyl ethers. Experiments at elevated temperatures indicate that thioglycolate attacks diethyl ether in water, in the absence of enzymes, with a rate constant of 6 × 10-11 M-1 s-1 at 25 °C and that ether thiolysis is strongly favored thermodynamically, with a Keq value of 2.5 × 106 (ΔG = -8.7 kcal/mol). Compared with the rate of non-enzymatic thiolysis, the lignin-degrading etherases LigE and LigF produce 1015-fold rate enhancements, among the largest that have been observed for an enzyme acting on two substrates.

Effects of Sepantronium Bromide (YM-155) on the Whole Transcriptome of MDA-MB-231 Cells: Highlight on Impaired ATR/ATM Fanconi Anemia DNA Damage Response.

Sepantronium bromide (YM-155) is believed to elicit apoptosis and mitotic arrest in tumor cells by reducing (BIRC5, survivin) mRNA. In this study, we monitored changes in survivin mRNA and protein after treating MDA-MB-231 cells with YM-155 concurrent with evaluation of whole transcriptomic (WT) mRNA and long intergenic non-coding RNA at 2 time points: 8 h sub-lethal (83 ng/mL) and 20 h at the LC50 (14.6 ng/mL). The data show a tight association between cell death and the precipitating loss of survivin protein and mRNA (-2.67 fold-change (FC), p<0.001) at 20 h, questioning if the decline in survivin is attributed to cell death or drug impact. The meager loss of survivin mRNA was overshadowed by enormous differential change to the WT in both magnitude and significance for over 2000 differentially up/down-regulated transcripts: (+22 FC to -12 FC, p<0.001). The data show YM-155 to up-regulate transcripts in control of circadian rhythm (NOCT, PER, BHLHe40, NFIL3), tumor suppression (SIK1, FOSB), histone methylation (KDM6B) and negative feedback of NF-kappa B signaling (TNFAIP3). Down-regulated transcripts by YM-155 include glucuronidase (GUSBP3), numerous micro-RNAs, DNA damage repair elements (CENPI, POLQ, RAD54B) and the most affected system was the ataxia-telangiectasia mutated (ATM)/Fanconi anemia E3 monoubiquitin ligase core complexes (FANC transcripts - A/B/E/F/G/M), FANC2, FANCI, BRCA1, BRCA2, RAD51, PALB2 gene and ATR (ATM- and Rad3-Related) pathway. In conclusion, these findings suggest that a primary target of YM-155 is the loss of replicative DNA repair systems.

Sulfonium Ion Condensation: The Burden Borne by SAM Synthetase.

S-Adenosylmethionine (SAM+) serves as the principal methylating agent in biological systems, but the thermodynamic basis of its reactivity does not seem to have been clearly established. Here, we show that methionine, methanol, and H+ combine to form S-methylmethionine (SMM+) with a temperature-independent equilibrium constant of 9.9 M-2. The corresponding group transfer potential of SMM+ (its free energy of hydrolysis at pH 7) is -8.2 kcal/mol. The "energy-rich" nature of sulfonium ions is related to the extreme acidity (p Ka -5.4) of the S-protonated thioether produced by sulfonium hydrolysis, and the large negative free energy of deprotonation of that species in neutral solution (-16.7 kcal/mol). At pH 7, SAM synthetase requires the free energy released by cleavage of two bonds of ATP to reverse that process.

Transcriptomic Profiling of MDA-MB-231 Cells Exposed to Boswellia Serrata and 3-O-Acetyl-B-Boswellic Acid; ER/UPR Mediated Programmed Cell Death.

BACKGROUND/AIM: Triple-negative breast cancer (TNBC) is characterized by the absence of hormone receptors (estrogen, progesterone and human epidermal growth factor receptor-2) and a relatively poor prognosis due to inefficacy of hormone receptor-based chemotherapies. It is imperative that we continue to explore natural products with potential to impede growth and metastasis of TNBC. In this study, we screened over 1,000 natural products for capacity to induce cell death in TNBC (MDA-MB -231) cells. MATERIALS AND METHODS: Frankincense (Boswellia serrata extract (BSE)) and 3-O-Acetyl-ß-boswellic acid (3-OAßBA) were relatively potent, findings that corroborate the body of existing literature. The effects of BSE and 3-OAßBA on genetic parameters in MDA-MB-231 cells were evaluated by examining whole-transcriptomic influence on mRNAs, long intergenic non-coding RNA transcripts (lincRNA) and non-coding miRNAs. RESULTS: Bio-statistical analysis demarcates the primary effect of both BSE/3-OAßBA on the up-regulation of PERK (protein kinase RNA-like endoplasmic reticulum kinase)- endoplasmic reticulum (ER)/unfolded protein response (UPR) pathways that are closely tied to activated programmed cell death (APCD). Global profiling confirms concomitant effects of BSE/3-OAßBA on upwardly expressed ER/URP APCD key components PERK (EIF2AK3), XBP1, C/EBP homologous protein transcription factor (CHOP), ATF3 and DDIT3,4/DNA-damage-inducible transcript 3,4 (GADD34). Further, BSE and/or 3-OAßBA significantly down-regulated oncogenes (OG) which, heretofore, lack functional pathway mapping, but are capable of driving epithelial-mesenchymal transition (EMT), cell survival, proliferation, metastasis and drug resistance. Among these are cell migration-inducing protein hyaluronan binding (CEMIP) [-7.22]; transglutaminase 2 [-4.96], SRY box 9 (SOX9) [-4.09], inhibitor of DNA binding 1, dominant negative helix-loop-helix protein (ID1) [-6.56]; and endothelin 1 (EDN1, [-5.06]). Likewise, in the opposite manner, BSE and/or 3-OAßBA induced the robust overexpression of tumor suppressor genes (TSGs), including: glutathione-depleting ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1) [+21.67]; the mTOR inhibitors - sestrin 2 (SESN2) [+16.4] Tribbles homolog 3 (TRIB3) [+6.2], homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERPUD1) [+12.01]; and cystathionine gamma-lyase (CTH) [+11.12]. CONCLUSION: The anti-cancer effects of the historically used frankincense sap (BSE) appear to involve major impact on the ER/UPR response, concomitant to effecting multiple targets counter to the growth, proliferation and metastasis of TNBC cancer cells. The microarray data are available at Expression Omnibus GEO Series accession number GSE102891.

Three Pyrimidine Decarboxylations in the Absence of a Catalyst.

The epigenetic modification of DNA by 5-methylation of cytosine residues can be reversed by the action of the TET family of dioxygenases that oxidize the methyl group to produce 5-carboxycytosine (5caC), which can be converted to cytosine in a final decarboxylation step. Likewise, 5-carboxyuracil (5caU) is decarboxylated to uracil in the last step in pyrimidine salvage. In view of the extreme difficulty of decarboxylating derivatives of orotic acid (6caU), it seemed desirable to establish the rates of decarboxylation of 5caC and 5caU in the absence of a catalyst. Arrhenius analysis of experiments performed at elevated temperatures indicates that 5caU decomposes with a rate constant of 1.1 × 10-9 s-1 (ΔH⧧ = 25 kcal/mol) in a neutral solution at 25 °C. The decomposition of 5caC is somewhat slower (k25 = 5.0 × 10-11 s-1; ΔH⧧ = 27 kcal/mol) and leads to the initial accumulation of cytosine as an intermediate, followed by the relatively rapid deamination of cytosine (k25 = 1.9 × 10-10 s-1; ΔH⧧ = 23.4 kcal/mol). Both 5caC and 5caU are decarboxylated many orders of magnitude more rapidly than 6caU is (k25 = 1.3 × 10-17 s-1). Ab initio simulations indicate that in all three cases, the favored route of spontaneous decarboxylation in water involves direct elimination of CO2 with the assistance of an explicit water molecule.

Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history.

The hydrolytic deamination of cytosine and 5-methylcytosine residues in DNA appears to contribute significantly to the appearance of spontaneous mutations in microorganisms and in human disease. In the present work, we examined the mechanism of cytosine deamination and the response of the uncatalyzed reaction to changing temperature. The positively charged 1,3-dimethylcytosinium ion was hydrolyzed at a rate similar to the rate of acid-catalyzed hydrolysis of 1-methylcytosine, for which it furnishes a satisfactory kinetic model and a probable mechanism. In agreement with earlier reports, uncatalyzed deamination was found to proceed at very similar rates for cytosine, 1-methylcytosine, cytidine, and cytidine 5'-phosphate, and also for cytosine residues in single-stranded DNA generated from a phagemid, in which we sequenced an insert representing the gene of the HIV-1 protease. Arrhenius plots for the uncatalyzed deamination of cytosine were linear over the temperature range from 90 °C to 200 °C and indicated a heat of activation (ΔH(‡)) of 23.4 ± 0.5 kcal/mol at pH 7. Recent evidence indicates that the surface of the earth has been cool enough to support life for more than 4 billion years and that life has been present for almost as long. If the temperature at Earth's surface is assumed to have followed Newton's law of cooling, declining exponentially from 100 °C to 25 °C during that period, then half of the cytosine-deaminating events per unit biomass would have taken place during the first 0.2 billion years, and <99.4% would have occurred during the first 2 billion years.

Temperature dependence of amino acid hydrophobicities.

The hydrophobicities of the 20 common amino acids are reflected in their tendencies to appear in interior positions in globular proteins and in deeply buried positions of membrane proteins. To determine whether these relationships might also have been valid in the warm surroundings where life may have originated, we examined the effect of temperature on the hydrophobicities of the amino acids as measured by the equilibrium constants for transfer of their side-chains from neutral solution to cyclohexane (K(w > c)). The hydrophobicities of most amino acids were found to increase with increasing temperature. Because that effect is more pronounced for the more polar amino acids, the numerical range of K(w > c) values decreases with increasing temperature. There are also modest changes in the ordering of the more polar amino acids. However, those changes are such that they would have tended to minimize the otherwise disruptive effects of a changing thermal environment on the evolution of protein structure. Earlier, the genetic code was found to be organized in such a way that--with a single exception (threonine)--the side-chain dichotomy polar/nonpolar matches the nucleic acid base dichotomy purine/pyrimidine at the second position of each coding triplet at 25 °C. That dichotomy is preserved at 100 °C. The accessible surface areas of amino acid side-chains in folded proteins are moderately correlated with hydrophobicity, but when free energies of vapor-to-cyclohexane transfer (corresponding to size) are taken into consideration, a closer relationship becomes apparent.

The nonenzymatic decomposition of guanidines and amidines.

To establish the rates and mechanisms of decomposition of guanidine and amidine derivatives in aqueous solution and the rate enhancements produced by the corresponding enzymes, we examined their rates of reaction at elevated temperatures and used the Arrhenius equation to extrapolate the results to room temperature. The similar reactivities of methylguanidine and 1,1,3,3-tetramethylguanidine and their negative entropies of activation imply that their decomposition proceeds by hydrolysis rather than elimination. The influence of changing pH on the rate of decomposition is consistent with attack by hydroxide ion on the methylguanidinium ion (k2 = 5 × 10(-6) M(-1) s(-1) at 25 °C) or with the kinetically equivalent attack by water on uncharged methylguanidine. At 25 °C and pH 7, N-methylguanidine is several orders of magnitude more stable than acetamidine, urea, or acetamide. Under the same conditions, the enzymes arginase and agmatinase accelerate substrate hydrolysis 4 × 10(14)-fold and 6 × 10(12)-fold, respectively, by mechanisms that appear to involve metal-mediated water attack. Arginine deiminase accelerates substrate hydrolysis 6 × 10(12)-fold by a mechanism that (in contrast to the mechanisms employed by arginase and agmatinase) is believed to involve attack by an active-site cysteine residue.

Amide bonds to the nitrogen atoms of cysteine and serine as "weak points" in the backbones of proteins.

During the initial event in protein self-splicing, a peptide bond to the nitrogen atom of an internal cysteine or serine residue is usually cleaved by the side chain -SH or -OH group to yield a thioester or oxyester intermediate that undergoes further reactions. Self-splicing reactions also accompany the maturation of hedgehog signaling proteins, plant-type asparaginases, and pyruvoyl enzymes. It would be of interest to know whether peptide bonds that involve the nitrogen atoms of cysteine or serine are more susceptible to cleavage than peptide bonds to amino acids that lack reactive side chains. Extrapolations of the results of model reactions conducted at elevated temperatures indicate that the -SH group of N-acetylcysteine enhances the rate of its hydrolysis by a factor of 70, while the OH group of N-acetylserine enhances the rate of its hydrolysis 12-fold, compared with the rate of hydrolysis of N-acetylalanine in neutral solution at 25 °C. Several lines of evidence suggest that the rate-enhancing effects of these -SH and -OH side chains arise from their ability to act as intramolecular general acid-base catalysts for hydrolysis, rather than as nucleophilic catalysts. The protein environment within self-splicing proteins appears to redirect the actions of these side chains to nucleophilic attack, generating rate enhancements that approach the rate enhancements generated by conventional enzymes.

Kinetic challenges facing oxalate, malonate, acetoacetate, and oxaloacetate decarboxylases.

To compare the powers of the corresponding enzymes as catalysts, the rates of uncatalyzed decarboxylation of several aliphatic acids (oxalate, malonate, acetoacetate, and oxaloacetate) were determined at elevated temperatures and extrapolated to 25 °C. In the extreme case of oxalate, the rate of the uncatalyzed reaction at pH 4.2 was 1.1 × 10(-12) s(-1), implying a 2.5 × 10(13)-fold rate enhancement by oxalate decarboxylase. Whereas the enzymatic decarboxylation of oxalate requires O(2) and Mn(II), the uncatalyzed reaction is unaffected by the presence of these cofactors and appears to proceed by heterolytic elimination of CO(2).

Impact of temperature on the time required for the establishment of primordial biochemistry, and for the evolution of enzymes.

All reactions are accelerated by an increase in temperature, but the magnitude of that effect on very slow reactions does not seem to have been fully appreciated. The hydrolysis of polysaccharides, for example, is accelerated 190,000-fold when the temperature is raised from 25 to 100 °C, while the rate of hydrolysis of phosphate monoester dianions increases 10,300,000-fold. Moreover, the slowest reactions tend to be the most heat-sensitive. These tendencies collapse, by as many as five orders of magnitude, the time that would have been required for early chemical evolution in a warm environment. We propose, further, that if the catalytic effect of a "proto-enzyme"--like that of modern enzymes--were mainly enthalpic, then the resulting rate enhancement would have increased automatically as the environment became cooler. Several powerful nonenzymatic catalysts of very slow biological reactions, notably pyridoxal phosphate and the ceric ion, are shown to meet that criterion. Taken together, these findings greatly reduce the time that would have been required for early chemical evolution, countering the view that not enough time has passed for life to have evolved to its present level of complexity.

Orotic acid decarboxylation in water and nonpolar solvents: a potential role for desolvation in the action of OMP decarboxylase.

OMP decarboxylase (ODCase) generates a very large rate enhancement without the assistance of metals or other cofactors. The uncatalyzed decarboxylation of 1-methylorotate in water is shown to involve the monoanion, although uncharged 1-methylorotic acid is decarboxylated at a similar rate. To measure the extent to which the rate of the nonenzymatic decarboxylation of orotate derivatives might be enhanced by their removal from solvent water, the 1-phosphoribosyl moiety of OMP was replaced with 1-substituents that would allow it to enter less polar solvents. When the tetrabutylammonium salt of 1-cyclohexylorotate was transferred from water to a series of dipolar aprotic solvents, its rate of decarboxylation increased markedly, varying with the relative ability of each solvent to release the substrate in the ground state from stabilization by solvent water acting as a proton donor. These findings are consistent with the view that separation of the substrate from solvent water may contribute, at least to a limited extent, to the rate enhancement produced by ODCase. This enzyme's active site, like that of another cofactorless enzyme recently shown to produce a rate enhancement similar in magnitude (uroporphyrinogen decarboxylase), is equipped with an ammonium group positioned in such a way as to balance the electrostatic charge of the carboxylate group of the substrate and later supply a proton to the incipient carbanion in a relatively waterless environment.

Uroporphyrinogen decarboxylation as a benchmark for the catalytic proficiency of enzymes.

The magnitude of an enzyme's affinity for the altered substrate in the transition state exceeds its affinity for the substrate in the ground state by a factor matching the rate enhancement that the enzyme produces. Particularly remarkable are those enzymes that act as simple protein catalysts, without the assistance of metals or other cofactors. To determine the extent to which one such enzyme, human uroporphyrinogen decarboxylase, enhances the rate of substrate decarboxylation, we examined the rate of spontaneous decarboxylation of pyrrolyl-3-acetate. Extrapolation of first-order rate constants measured at elevated temperatures indicates that this reaction proceeds with a half-life of 2.3 x 10(9) years at 25 degrees C in the absence of enzyme. This enzyme shows no significant homology with orotidine 5'-monophosphate decarboxylase (ODCase), another cofactorless enzyme that catalyzes a very slow reaction. It is proposed that, in both cases, a protonated basic residue (Arg-37 in the case of human UroD; Lys-93 in the case of yeast ODCase) furnishes a counterion that helps the scissile carboxylate group of the substrate leave water and enter a relatively nonpolar environment, stabilizes the incipient carbanion generated by the departure of CO(2), and supplies the proton that takes its place.

Indiscriminate binding by orotidine 5'-phosphate decarboxylase of uridine 5'-phosphate derivatives with bulky anionic c6 substituents.

Orotidine 5'-phosphate (OMP) decarboxylase appears to act upon its substrate without the intervention of metals or other cofactors and without the formation of covalent bonds between the enzyme and the substrate. Crystallographic information indicates that substrate binding forces the substrate's scissile carboxylate group into the neighborhood of several charged groups at the active site. It has been proposed that binding might result in electrostatic stress at the substrate's C6 carboxylate group in such a way as to promote decarboxylation by destabilizing the enzyme-substrate complex in its ground state. If that were the case, one would expect uridine 5'-phosphate (UMP) derivatives with bulky anionic substituents at C6 to be bound weakly compared with UMP, which is unsubstituted at C6. Here, we describe the formation of anionic 5,6-dihydro-6-sulfonyl derivatives by spontaneous addition of sulfite to UMP and to OMP. These sulfite addition reactions, which are slowly reversible and are not catalyzed by the enzyme, result in the appearance of one (or, in the case of OMP, two) bulky anionic substituents at the 6-carbon atom of UMP. These inhibitors are bound with affinities that surpass the binding affinity of UMP. We are led to infer that the active site of OMP decarboxylase is remarkably accommodating in the neighborhood of C6. These are not the properties that one would expect of an active site with a rigid structure that imposes sufficient electrostatic stress on the substrate to produce a major advancement along the reaction coordinate.