Use of pH and kinetic isotope effects to establish chemistry as rate-limiting in oxidation of a peptide substrate by LSD1.

The mechanism of oxidation of a peptide substrate by the flavoprotein lysine-specific demethylase (LSD1) has been examined using the effects of pH and isotopic substitution on steady-state and rapid-reaction kinetic parameters. The substrate contained the 21 N-terminal residues of histone H3, with a dimethylated lysyl residue at position 4. At pH 7.5, the rate constant for flavin reduction, k(red), equals k(cat), establishing the reductive half-reaction as rate-limiting at physiological pH. Deuteration of the lysyl methyls results in identical kinetic isotope effects of 3.1 +/- 0.2 on the k(red), k(cat), and k(cat)/K(m) values for the peptide, establishing C-H bond cleavage as rate-limiting with this substrate. No intermediates between oxidized and reduced flavin can be detected by stopped-flow spectroscopy, consistent with the expectation for a direct hydride transfer mechanism. The k(cat)/K(m) value for the peptide is bell-shaped, consistent with a requirement that the nitrogen at the site of oxidation be uncharged and that at least one of the other lysyl residues be charged for catalysis. The (D)(k(cat)/K(m)) value for the peptide is pH-independent, suggesting that the observed value is the intrinsic deuterium kinetic isotope effect for oxidation of this substrate.

Facile synthesis of substituted trans-2-arylcyclopropylamine inhibitors of the human histone demethylase LSD1 and monoamine oxidases A and B.

A facile synthetic route to substituted trans-2-arylcyclopropylamines was developed to provide access to mechanism-based inhibitors of the human flavoenzyme oxidase lysine-specific histone demethylase LSD1 and related enzyme family members such as monoamine oxidases A and B.

trans-2-Phenylcyclopropylamine is a mechanism-based inactivator of the histone demethylase LSD1.

The catalytic domain of the flavin-dependent human histone demethylase lysine-specific demethylase 1 (LSD1) belongs to the family of amine oxidases including polyamine oxidase and monoamine oxidase (MAO). We previously assessed monoamine oxidase inhibitors (MAOIs) for their ability to inhibit the reaction catalyzed by LSD1 [Lee, M. G., et al. (2006) Chem. Biol. 13, 563-567], demonstrating that trans-2-phenylcyclopropylamine (2-PCPA, tranylcypromine, Parnate) was the most potent with respect to LSD1. Here we show that 2-PCPA is a time-dependent, mechanism-based irreversible inhibitor of LSD1 with a KI of 242 microM and a kinact of 0.0106 s-1. 2-PCPA shows limited selectivity for human MAOs versus LSD1, with kinact/KI values only 16-fold and 2.4-fold higher for MAO B and MAO A, respectively. Profiles of LSD1 activity and inactivation by 2-PCPA as a function of pH are consistent with a mechanism of inactivation dependent upon enzyme catalysis. Mass spectrometry supports a role for FAD as the site of covalent modification by 2-PCPA. These results will provide a foundation for the design of cyclopropylamine-based inhibitors that are selective for LSD1 to probe its role in vivo.

Histone H3 lysine 4 demethylation is a target of nonselective antidepressive medications.

Demethylation of histone H3 lysine 4 is carried out by BHC110/LSD1, an enzyme with close homology to monoamine oxidases (MAO). Monoamine oxidase A or B are frequent targets of selective and nonselective small molecular inhibitors used for treatment of depression. Here we show that in contrast to selective monoamine oxidase inhibitors such as pargyline, nonselective monoamine oxidase inhibitors potently inhibit nucleosomal demethylation of histone H3 lysine 4. Tranylcypromine (brand name Parnate) displayed the best inhibitory activity with an IC50 of less than 2 microM. Treatment of P19 embryonal carcinoma cells with tranylcypromine resulted in global increase in H3K4 methylation as well as transcriptional derepression of two BHC110 target genes, Egr1 and the pluripotent stem cell marker Oct4. These results attest to the effectiveness of tranylcypromine as a small molecular inhibitor of histone demethylation.

Evolutionary potential of (beta/alpha)8-barrels: in vitro enhancement of a "new" reaction in the enolase superfamily.

The repertoire of reactions in the mechanistically diverse enolase superfamily is the result of divergent evolution that conserved enolization of a carboxylate anion substrate but allowed different overall reactions using different substrates. Details of the pathways for the natural evolutionary process are unknown, but the events reasonably involve (1) incremental increases in the level of the "new" reaction that would provide a selective advantage and (2) an accompanying loss of the "old" reaction catalyzed by the progenitor. In an effort to better understand the molecular processes of divergent evolution, the D297G mutant of the l-Ala-d/l-Glu epimerase (AEE) from Escherichia coli was designed so that it could bind the substrate for the o-succinylbenzoate synthase (OSBS) reaction and, as a result, catalyze that reaction [Schmidt, D. M. Z., Mundorff, E. C., Dojka, M., Bermudez, E., Ness, J. E., Govindarajan, S., Babbitt, P. C., Minshull, J., and Gerlt, J. A. (2003) Biochemistry 42, 8387-8393]. The AEE progenitor did not catalyze the OSBS reaction, but the D297G mutant catalyzed a low level of the OSBS reaction (k(cat), 0.013 s(-)(1); K(m), 1.8 mM; k(cat)/K(m), 7.4 M(-)(1) s(-)(1)) that was sufficient to permit anaerobic growth by an OSBS-deficient strain of E. coli; the level of the progenitor's natural AEE reaction was significantly diminished. Using random mutagenesis and an anaerobic metabolic selection, we now have identified the I19F substitution as an additional mutation that enhances both growth of the OSBS-deficient strain and the kinetic constants for the OSBS reaction (k(cat), 0.031 s(-)(1); K(m), 0.34 mM; k(cat)/K(m), 90 M(-)(1) s(-)(1)). Several other substitutions for Ile 19 also enhanced the level of the OSBS reaction. All of the substitutions substantially decreased the level of the AEE reaction from that possessed by the D297G progenitor. The changes in the kinetic constants for both the OSBS and AEE reactions are attributed to a readjustment of substrate specificity so that the substrate for the OSBS reaction is more productively presented to the conserved acid/base catalysts in the active site. These observations support our hypothesis that evolution of "new" functions in the enolase superfamily can occur simply by changes in specificity-determining residues.

Evolution of enzymatic activities in the enolase superfamily: structure of a substrate-liganded complex of the L-Ala-D/L-Glu epimerase from Bacillus subtilis.

The members of the mechanistically diverse enolase superfamily share a bidomain structure formed from a (beta/alpha)7beta-barrel domain [a modified (beta/alpha)8- or TIM-barrel] and a capping domain formed from N- and C-terminal segments of the polypeptide. The active sites are located at the interface between the C-terminal ends of the beta-strands in the barrel domain and two flexible loops in the capping domain. Within this structure, the acid/base chemistry responsible for formation and stabilization of an enediolate intermediate derived from a carboxylate anion substrate and the processing of it to product is "hard-wired" by functional groups at the C-terminal ends of the beta-strands in the barrel domain; the identity of the substrate is determined in part by the identities of residues located at the end of the eighth beta-strand in the barrel domain and two mobile loops in the capping domain. On the basis of the identities of the acid/base functional groups at the ends of the beta-strands, the currently available structure-function relationships derived from functionally characterized members are often sufficient for "deciphering" the identity of the chemical reaction catalyzed by sequence-divergent members discovered in genome projects. However, insufficient structural information for liganded complexes for specifying the identity of the substrate is available. In this paper, the structure of the complex of L-Ala-L-Glu with the L-Ala-D/L-Glu epimerase from Bacillus subtilis is reported. As expected for the 1,1-proton transfer reaction catalyzed by this enzyme, the alpha-carbon of the substrate is located between Lys 162 and Lys 268 at the ends of the second and sixth beta-strands in the barrel domain. The alpha-ammonium group of the l-Ala moiety is hydrogen bonded to both Asp 321 and Asp 323 at the end of the eighth beta-strand, revealing a novel strategy for substrate recognition in the superfamily. The delta-carboxylate group of the Glu moiety is hydrogen bonded to Arg 24 in one of the flexible loops in the capping domain, thereby providing a structural explanation for the restricted substrate specificity of this epimerase [Schmidt, D. M., Hubbard, B. K., and Gerlt, J. A. (2001) Biochemistry 40, 15707-15715]. These studies provide important new information about the structural bases for substrate specificity in the enolase superfamily.

Chemical and enzymatic synthesis of fluorinated-dehydroalanine-containing peptides.

Michael acceptors have long been recognized as reactive functionalities that may link a biologically active molecule to its cellular target. 1,2-Dehydro amino acids are potential Michael acceptors present in a large number of natural products, but their reactivity is modulated by the deactivating nature of the alpha-amino group engaged in an amide bond. We describe here the preparation of 3-fluoro-1,2-dehydroalanine moieties within peptides that significantly enhance the reactivity of the Michael acceptor. Two different routes were designed to access these compounds, one relying on chemical means to introduce the desired functionality and the second taking advantage of a peptide epimerase. In the chemical approach, the fluoro-Pummerer reaction of cysteine derivatives afforded 3-fluorocysteine residues that were oxidized to the corresponding sulfoxides, followed by thermolytic elimination to provide the desired 3-fluorodehydroalanines. The mechanism of the fluoro-Pummerer reaction was investigated and several possible pathways were ruled out. The enzymatic approach utilized the dipeptide epimerase YcjG from Escherichia coli. Difluorinated alanine was incorporated at the C terminus of a dipeptide by chemical means. The resulting peptide proved to be a substrate for YcjG, which catalyzed fluoride elimination to provide the 3-fluorodehydroalanine-containing peptide. Mechanistic investigations showed that fluoride elimination occurred faster than epimerization and at a rate close to that of epimerization of Ala-Ala.

Evolutionary potential of (beta/alpha)8-barrels: functional promiscuity produced by single substitutions in the enolase superfamily.

The members of the mechanistically diverse, (beta/alpha)(8)-barrel fold-containing enolase superfamily evolved from a common progenitor but catalyze different reactions using a conserved partial reaction. The molecular pathway for natural divergent evolution of function in the superfamily is unknown. We have identified single-site mutants of the (beta/alpha)(8)-barrel domains in both the l-Ala-d/l-Glu epimerase from Escherichia coli (AEE) and the muconate lactonizing enzyme II from Pseudomonas sp. P51 (MLE II) that catalyze the o-succinylbenzoate synthase (OSBS) reaction as well as the wild-type reaction. These enzymes are members of the MLE subgroup of the superfamily, share conserved lysines on opposite sides of their active sites, but catalyze acid- and base-mediated reactions with different mechanisms. A comparison of the structures of AEE and the OSBS from E. coli was used to design the D297G mutant of AEE; the E323G mutant of MLE II was isolated from directed evolution experiments. Although neither wild-type enzyme catalyzes the OSBS reaction, both mutants complement an E. coli OSBS auxotroph and have measurable levels of OSBS activity. The analogous mutations in the D297G mutant of AEE and the E323G mutant of MLE II are each located at the end of the eighth beta-strand of the (beta/alpha)(8)-barrel and alter the ability of AEE and MLE II to bind the substrate of the OSBS reaction. The substitutions relax the substrate specificity, thereby allowing catalysis of the mechanistically diverse OSBS reaction with the assistance of the active site lysines. The generation of functionally promiscuous and mechanistically diverse enzymes via single-amino acid substitutions likely mimics the natural divergent evolution of enzymatic activities and also highlights the utility of the (beta/alpha)(8)-barrel as a scaffold for new function.