3106-60-3Relevant academic research and scientific papers
Development of a Suicide Inhibition-Based Protein Labeling Strategy for Nicotinamide N-Methyltransferase
Sen, Sudeshna,Mondal, Santanu,Zheng, Li,Salinger, Ari J.,Fast, Walter,Weerapana, Eranthie,Thompson, Paul R.
, p. 613 - 618 (2019)
Nicotinamide N-methyltransferase (NNMT) catalyzes the S-adenosyl-l-methionine-dependent methylation of nicotinamide to form N-methylnicotinamide. This enzyme detoxifies xenobiotics and regulates NAD+ biosynthesis. Additionally, NNMT is overexpressed in various cancers. Herein, we describe the first NNMT-targeted suicide substrates. These compounds, which include 4-chloropyridine and 4-chloronicotinamide, exploit the broad substrate scope of NNMT; methylation of the pyridine nitrogen enhances the electrophilicity of the C4 position, thereby promoting an aromatic nucleophilic substitution by C159, a noncatalytic cysteine. On the basis of this activity, we developed a suicide inhibition-based protein labeling strategy using an alkyne-substituted 4-chloropyridine that selectively labels NNMT in vitro and in cells. In total, this study describes the first NNMT-directed activity-based probes.
Structural basis of substrate recognition in human nicotinamide N-methyltransferase
Peng, Yi,Sartini, Davide,Pozzi, Valentina,Wilk, Dennis,Emanuelli, Monica,Yee, Vivien C.
, p. 7800 - 7808 (2011)
Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridines, and other analogues using S-adenosyl-l-methionine as donor. NNMT plays a significant role in the regulation of metabolic pathways and is expressed at markedly high levels in several kinds of cancers, presenting it as a potential molecular target for cancer therapy. We have determined the crystal structure of human NNMT as a ternary complex bound to both the demethylated donor S-adenosyl-l-homocysteine and the acceptor substrate nicotinamide, to 2.7 A resolution. These studies reveal the structural basis for nicotinamide binding and highlight several residues in the active site which may play roles in nicotinamide recognition and NNMT catalysis. The functional importance of these residues was probed by mutagenesis. Of three residues near the nicotinamide's amide group, substitution of S201 and S213 had no effect on enzyme activity while replacement of D197 dramatically decreased activity. Substitutions of Y20, whose side chain hydroxyl interacts with both the nicotinamide aromatic ring and AdoHcy carboxylate, also compromised activity. Enzyme kinetics analysis revealed kcat/Km decreases of 2-3 orders of magnitude for the D197A and Y20A mutants, confirming the functional importance of these active site residues. The mutants exhibited substantially increased Km for both NCA and AdoMet and modestly decreased kcat. MD simulations revealed long-range conformational effects which provide an explanation for the large increase in K m(AdoMet) for the D197A mutant, which interacts directly only with nicotinamide in the ternary complex crystal structure.
MASS SPECTROMETRY OF NITROGEN HETEROCYCLES. 2. CORRELATION OF MASS SPECTROMETRIC FRAGMENTATION PROCESSES AND CHEMICAL AROMATIZATION REACTIONS OF DIHYDROAZINES
Chupakhin, O. N.,Baklykov, V. G.,Klyuev, N. A.,Matern, A. I.
, p. 904 - 907 (1989)
The results of mass spectrometric studies of aromatization processes of dihydroazines have been compared with experimental data relative to their chemical oxidation.The results of electron impact induced ionization of dihydroazines can be correlated direc
A Rapid and Efficient Assay for the Characterization of Substrates and Inhibitors of Nicotinamide N-Methyltransferase
Van Haren, Matthijs J.,Sastre Tora?o, Javier,Sartini, Davide,Emanuelli, Monica,Parsons, Richard B.,Martin, Nathaniel I.
, p. 5307 - 5315 (2016/09/28)
Nicotinamide N-methyltransferase (NNMT) is one of the most abundant small molecule methyltransferases in the human body and is primarily responsible for the N-methylation of the nicotinamide (vitamin B3). Employing the cofactor S-adenosyl-l-methionine, NNMT transfers a methyl group to the pyridine nitrogen of nicotinamide to generate N-methylnicotinamide. Interestingly, NNMT is also able to N-methylate a variety of other pyridine-containing small molecules, suggesting a secondary role for the enzyme in the detoxification of xenobiotics. A number of recent studies have also revealed links between NNMT overexpression and a variety of diseases, including multiple cancers, Parkinson's disease, diabetes, and obesity. To facilitate further study of both the substrate scope and potential for inhibitor development, we here describe the development of a new NNMT activity assay. The assay makes use of ultra-high-performance hydrophilic interaction chromatography, allowing for rapid separation of the reaction products, coupled with quadrupole time-of-flight mass spectrometric detection, providing for enhanced sensitivity and enabling high-throughput sample analysis. We successfully demonstrated the general applicability of the method by performing kinetic analyses of NNMT-mediated methylation for a range of pyridine-based substrates. These findings also provide new insight into the diversity of substrate recognition by NNMT in a quantitative manner. In addition, we further established the suitability of the assay for the identification and characterization of small molecule inhibitors of NNMT. To do so, we investigated the inhibition of NNMT by the nonspecific methyltransferase inhibitors sinefungin and S-adenosyl-l-homocysteine, revealing IC50 values in the low micromolar range. The results of these inhibition studies are particularly noteworthy as they will permit future efforts toward the development of new NNMT-specific inhibitors.
A classical but new kinetic equation for hydride transfer reactions
Zhu, Xiao-Qing,Deng, Fei-Huang,Yang, Jin-Dong,Li, Xiu-Tao,Chen, Qiang,Lei, Nan-Ping,Meng, Fan-Kun,Zhao, Xiao-Peng,Han, Su-Hui,Hao, Er-Jun,Mu, Yuan-Yuan
, p. 6071 - 6089 (2013/09/12)
A classical but new kinetic equation to estimate activation energies of various hydride transfer reactions was developed according to transition state theory using the Morse-type free energy curves of hydride donors to release a hydride anion and hydride acceptors to capture a hydride anion and by which the activation energies of 187 typical hydride self-exchange reactions and more than thirty thousand hydride cross transfer reactions in acetonitrile were safely estimated in this work. Since the development of the kinetic equation is only on the basis of the related chemical bond changes of the hydride transfer reactants, the kinetic equation should be also suitable for proton transfer reactions, hydrogen atom transfer reactions and all the other chemical reactions involved with breaking and formation of chemical bonds. One of the most important contributions of this work is to have achieved the perfect unity of the kinetic equation and thermodynamic equation for hydride transfer reactions. The Royal Society of Chemistry.
Characterization of nicotinamidases: Steady state kinetic parameters, classwide inhibition by nicotinaldehydes, and catalytic mechanism
French, Jarrod B.,Cen, Yana,Vrablik, Tracy L.,Xu, Ping,Allen, Eleanor,Hanna-Rose, Wendy,Sauve, Anthony A.
experimental part, p. 10421 - 10439 (2011/10/07)
Nicotinamidases are metabolic enzymes that hydrolyze nicotinamide to nicotinic acid. These enzymes are widely distributed across biology, with examples found encoded in the genomes of Mycobacteria, Archaea, Eubacteria, Protozoa, yeast, and invertebrates, but there are none found in mammals. Although recent structural work has improved our understanding of these enzymes, their catalytic mechanism is still not well understood. Recent data show that nicotinamidases are required for the growth and virulence of several pathogenic microbes. The enzymes of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans regulate life span in their respective organisms, consistent with proposed roles in the regulation of NAD+ metabolism and organismal aging. In this work, the steady state kinetic parameters of nicotinamidase enzymes from C. elegans, Sa. cerevisiae, Streptococcus pneumoniae (a pathogen responsible for human pneumonia), Borrelia burgdorferi (the pathogen that causes Lyme disease), and Plasmodium falciparum (responsible for most human malaria) are reported. Nicotinamidases are generally efficient catalysts with steady state kcat values typically exceeding 1 s -1. The Km values for nicotinamide are low and in the range of 2 -110 μM. Nicotinaldehyde was determined to be a potent competitive inhibitor of these enzymes, binding in the low micromolar to low nanomolar range for all nicotinamidases tested. A variety of nicotinaldehyde derivatives were synthesized and evaluated as inhibitors in kinetic assays. Inhibitions are consistent with reaction of the universally conserved catalytic Cys on each enzyme with the aldehyde carbonyl carbon to form a thiohemiacetal complex that is stabilized by a conserved oxyanion hole. The S. pneumoniae nicotinamidase can catalyze exchange of 18O into the carboxy oxygens of nicotinic acid with H218O. The collected data, along with kinetic analysis of several mutants, allowed us to propose a catalytic mechanism that explains nicotinamidase and nicotinic acid 18O exchange chemistry for the S. pneumoniae enzyme involving key catalytic residues, a catalytic transition metal ion, and the intermediacy of a thioester intermediate.
Direct observation of NADH radical cation generated in reactions with one-electron oxidants
Zielonka, Jacek,Marcinek, Andrzej,Adamus, Jan,Gebicki, Jerzy
, p. 9860 - 9864 (2007/10/03)
The formation of NADH radical cation in reactions with one-electron oxidants was observed for the first time. Transient products involving two tautomeric (keto and enol) forms of radical cation and neutral radical were spectroscopically characterized by means of pulse radiolysis. The kinetics of the decay of keto radical cation and neutral radical was investigated. The pKa value of the enol form of NADH radical cation was determined. The simple analogues of NADH and NAD+, namely, 1-methyl-1,4-dihydronicotinamide and it oxidized form, were studied for comparison.
Deuterium Isotope Effects for the Nonenzymatic and Glutamate Dehydrogenase Catalyzed Reduction of an α-Imino Acid by NADH
Srinivasan, R.,Fisher, Harvey F.
, p. 4301 - 4305 (2007/10/02)
The mechanisms of the nonenzymatic and glutamate dehydrogenase catalyzed reduction of an α-imino acid, Δ1-pyrroline-2-carboxylic acid, by NAD(P)H have been studied by deuterium isotope effects.The partition isotope effects for the nonenzymatic reaction with 4-deuterated 1,4-dihydronicotinamides are about the same as the corresponding observed kinetic isotope effects with 4,4-dideuterio-1,4-dihydronicotinamides, suggesting that the hydrogen-transfer step is solely rate limiting.This reaction is characterized by an intrinsic primary kinetic isotope effect of 1.3 and a very product-like transition state.The enzymatic reaction has been studied by determining the second-order rate constants for the reduction of the imino acid by the enzyme-NADH complex with 4,4-dideuterio and stereospecifically labeled 4-deuterio NADH.The primary isotope effect when the in-place hydrogen is protium is 3.80, and the secondary isotope effect when the in-flight hydrogen is protium is 1.21.Deuteration at one site lowers the isotope effects at the other by 13percent.The following conclusions emerge for the reduction of the imino acid by the enzyme-NADH complex: (1) the hydrogen-transfer step is at least rate contributing, (2) the transition state for this reaction is more symmetric than that of the nonenzymatic reaction, (3) both the C-4 hydrogens of NADH participate in the reaction coordinate motion, and (4) there is some nuclear tunneling in the reaction coordinate.The kinetic isotope effect for the oxidation of proline and proline-2-d by enzyme-NADP(+) is 4.1.
Radical Cations of some Low-potential Viologen Compounds; Reduction Potentials and Electron-transfer Reactions
Anderson, Robert F.,Patel, Kantilal B.
, p. 2693 - 2702 (2007/10/02)
The one-electron reduction potentials (E1) of certain pyrazinediium, diazepinediium and diazocinediium viologen compounds substituted with methyl groups, V(2+), have been determined from the position of the one-electron transfer equilibria with reference compounds using pulse radiolysis.E1 ranges from -491 +/- 6 mV (vs NHE) for 6,7-dihydro-2,11-dimethyldipyridopyrazinediium dibromide (V21(2+)) to -832 +/- 11 mV for 6,7,8,9-tetrahydro-2,3,12,13-tetramethyldipyridodiazocinediium dibromide (V42(2+)).The rates of reduction by e(1-)aq were found to be independent of E1 whereas the rates of electron transfer from CO2(1-). and (CH3)2C.OH species do show a dependence on E1 for V(2+) compounds of lowest E1.Marcus-type treatment of the rate-constant data yields a rate constant of electron exchange between propan-2-oxyl radicals and acetone in the range (2-6)E6 dm3mol-1s-1 and between the radical cation of 6,7-dihydro-2,3,10,11-tetramethyldipyridopyrazinediium dibromide (V22.(1+)) and its unreduced form (V22(2+)) of 5E7 dm3mol-1s-1.
The Pyridinium-Dihydropyridine System. Reduction Potentials and the Mechanism of Oxidation of 1,4-Dihydropyridines by a Shiff Base
Srinivasan, R.,Medary, Richard T.,Fisher, Harvey F.,Norris, Donald J.,Stewart, Ross
, p. 807 - 812 (2007/10/02)
As a model system for the glutamate dehydrogenase catalyzed reductive amination of α-ketoglutarate we have studied the reduction of a Shiff base, Δ1-pyrroline-2-carboxylic acid, by a series of 14 N-1 and C-3 substituted 1,4-dihydropyridines, including NMNH, NADH, and NADPH.The reversible electrode potentials of eight of the dihydropyridines, all dihydronicotinamides, have also been determined.The reduction reaction has the following characteristics: (a) it is first order in protonated Schiff base (zwitterionic form) and first order in the dihydropyridine, (b) there is a small deuterium isotope effect when the C-4 position of the dihydropyridine is deuterated (1.20-1.57 at 25 deg C), (c) there is a direct transfer of hydrogen from C-4 of the dihydropyridine to C-2 of the pyrroline, (d) the rates for seven N-1 substituted dihydronicotinamides are correlated satisfactorily with ?* giving ρ* = -1.98 (H2O) and -1.79 (aqueous methanol), there being only a modest difference in rates in these two solvents, (e) there is a good correlation between the rates of reduction by the dihydronicotinamides and the E0 values of the reversible two-electron dihydropyridine-pyridinium couple, the effect being 31.0 mV per logarithmic unit of rate, (f) there is a close correlation between the rates of reduction of pyrroline and of flavin by the dihydropyridines, and (g) the enthalpy and entropy of activation for the rate-controlling step in the reduction by 1-benzyl-1,4-dihydronicotinamide are 15.7 kcal mol-1 and -7.6 eu.We believe that direct hydride transfer has taken place to produce proline in a single step and it can be inferred that the transition state closely resembles products in structure.The similarity between pyrroline and flavin reduction suggests that the latter reaction may also be a direct hydride transfer.
