Progress 01/01/03 to 09/30/11
Outputs
OUTPUTS: Phosphoenolpyruvate carboxy kinase (PEPCK) is an enzyme involved in gluconeogenesis and is therefore important in glucose homeostasis. We have carried out research on the roles of metal ions in reaction catalyzed by PEPCK from Saccharomyces cerevisiae. Laccases are oxidases that contain multiple copper ions to carry out the oxidation of organic substrates such as ligninols with concomitant reduction of molecular oxygen to water. The laccases from plants are involved in synthesis of the biopolymer, lignin, as well as in wound healing. Fungal laccases are believed to be involved in degradation of lignin. We have carried out studies of the capacity of a laccase, from Trametes versicolor to carry out a one-electron oxidation of a Co(II) complex of ethylenediamine tetraacetic acid as a model of laccase mediated oxidations. PARTICIPANTS: Carolina Sepulveda, Fulbright Scholar, graduate student from University of Santiago, Chile; Scott Klasek, undergraduate UW-Madison, received training. TARGET AUDIENCES: The target audiences are the biochemical, biophysical, biological, and chemical research communities with the intent of increasing basic scientific knowledge regarding free radicals in biological systems. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Free radicals are important intermediates in many enzymatic reactions. Radicals are also potentially harmful by-products of oxidative metabolism. The transient nature of these reactive species requires special techniques for their characterization. Spectroscopic methods coupled with transient kinetic techniques such as rapid mix freeze quench trapping allows observation and characterization of these species. Our research is providing information that is crucial to understanding the biochemistry and biology of free radical reactions.
Publications
- Frey, P. A., Reed, G. H., Pyridoxal-5-phosphate as the catalyst for radical isomerization in reactions of PLP-dependent aminomutases, BBA (2011).
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Ethanolamine ammonia-lyase (EAL) is an adenosylcobalamin (AdoCbl) dependent enzyme that permits enteric bacteria to metabolize ethanolamine generated in the breakdown of phospholipids. The enzyme uses the AdoCbl cofactor to initiate a free radical reaction by which ethanolamine is converted to acetaldehyde and ammonia. The AdoCbl cofactor reforms at the end of each catalytic cycle as 5'-deoxyadenosine is re-converted to the 5'-yl radical by reaction with a product radical, 1-aminoethanol-2-yl. The product radical is too unstable to be trapped and observed by electron paramagnetic resonance (EPR) spectroscopy. We have characterized the suicide inactivation of EAL by acetamide-a structural analog of the resumed product, 1-aminoethanol. Rapid mix freeze quench methods showed that within the first 50 ms of mixing acetamide with the EAL-AdoCbl complex, the methylene radical of acetamide appears. The acetamide radical is spin coupled to Co2+ in the cob(II)alamin fragment of the cofactor. The identity of this acetamide radical was confirmed by using acetamide-d3. Computer simulations were used to extract the electron spin-spin and electron spin nuclear hyperfine parameters. Analysis of the radical-Co2+ coupling indicated that the radical center on C2 of acetamide was 9.8 Angstroms from Co2+. After longer incubation times, the methylene radical of acetamide gives way to a protein radical greater than 20 Angstroms from Co2+. The side chain of Val 192 is a strong candidate for the protein radical. Inactivation of the enzyme occurs because migration of the radical center out of the active site leads to permanent cleavage of AdoCbl and consequent blocking of the active site by cob(II)alamin. PARTICIPANTS: Russell Poyner, Associate Scientist; Ann Menefee, Research Assistant. Ann was a graduate student in the Biochemistry degree program, and therefore received research training. TARGET AUDIENCES: The target audiences are the biochemical, biophysical, biological, and chemical research communities with the intent of increasing basic scientific knowledge regarding free radicals in biological systems. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Free radicals are important intermediates in many enzymatic reactions. Radicals are also potentially harmful by-products of oxidative metabolism. The transient nature of these reactive species requires special techniques for their characterization. Spectroscopic methods coupled with transient kinetic techniques such as rapid mix freeze quench trapping allows observation and characterization of these species. Our research is providing information that is crucial to understanding the biochemistry and biology of free radical reactions.
Publications
- Tang, K.-H. , Mansoorabadi, S.O., Reed, G. H., Frey, P. A., Radical Triplets and Suicide Inhibition in Reactions of 4-Thia-D- and 4-Thia-L-lysine with Lysine 5,6-Aminomutase, Biochemistry 48, 8151-8160 (2009).
- Menefee, A. L., Product and Substrate Analogs in Mechanism-Based Inactivation of Ethanolamine Ammonia-Lyase, Ph. D. dissertation, University of Wisconsin, Dec. 2009.
- Martinez-Gomez, N. C., Poyner, R. R., Mansoorabadi, S. O., Reed, G. H., and Downs, D. M., Reaction of AdoMet with ThiC Generates a Backbone Free Radical, Biochemistry 48, 217-219 (2009).
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: We have used rapid mix freeze quench kinetic methods to trap the steady-state radical intermediate in the free radical mediated deamination of ethanolamine catalyzed by the adenosylcobalamin-dependent enzyme, ethanolamine ammonia-lyase. We used specifically 13C labeled forms of ethanolamine in combination with electron paramagnetic resonance (EPR) to determine that the substrate radical formed by hydrogen atom abstraction from C1 of the substrate was the only radical to achieve significant concentrations during the time course of turnover. We used isotopomers of ethanolamine with specific labeling with 13C, 15N, 2H, 17O to determine the conformation of the radical and the extent of spin delocalization. Computer simulations were used to evaluate the electron spin-spin interaction between the radical and the paramagnetic cobalt (II) of cob(II)alamin in the active site. Electronic structure calculations were used to assign the conformation of the radical and explore energetic feasiblity of various routes to carbon-nitroen bond breaking at carbon 2. We have also used EPR to examine the structure of two radicals formed in the oxidative decarboxylation of pyruvate by the thiamin pyrophosphate and iron-sulfur enzyme, pyruvate ferredoxin oxidoreductase (PFOR). We have used the EPR spectroscopic results as a basis for a new ,stepwise , oxidation sequence for PFOR. PARTICIPANTS: Russell Poyner Associate Scientist; Gunes Bender, Research Assistant; Ann Menefee, Research Assistant. Ann and Gunes are graduate students in the Biochemistry and Biophysics degree programs respectively and are therefore receiving research training. Kenneth C.Johnson is a part-time instrumentation specialist. TARGET AUDIENCES: The target audiences would be the biochemical, biophysical, and chemical scientific research communities with the intent of increasing knowledge. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Radicals are important intermediates in many enzymatic reactions as well as potentially harmful by-products of metabolism. These species are typically transient and uniquely suited for spectroscopic investigation. Our methods are revealing the structures of these naturally occurring species and the steps in their generation and propagation. These results are useful in understanding the biochemistry and biology of free radicals.
Publications
- Bender, G., Poyner, R. R., and Reed, G. H., Identification of the Substrate Radical Intermediate Derived from Ethanolamine During Catalysis by Ethanolamine Ammonia-Lyase, Biochemistry 47, 11360-11366 (2008).
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: We have used rapid mix freeze quench kinetic methods to trap the steady-state radical intermediate in the free radical mediated deamination of ethanolamine catalyzed by the adenosylcobalamin-dependent enzyme, ethanolamine ammonia-lyase. We used specifically 13C labeled forms of ethanolamine in combination with electron paramagnetic resonance (EPR) to determine that the substrate radical formed by hydrogen atom abstraction from C1 of the substrate was the only radical to achieve significant concentrations during the time course of turnover. We used isotopomers of ethanolamine with specific labeling with 13C, 15N, 2H, 17O to determine the conformation of the radical and the extent of spin delocalization. Computer simulations were used to evaluate the electron spin-spin interaction between the radical and the paramagnetic cobalt (II) of cob(II)alamin in the active site. Electronic structure calculations were used to assign the conformation of the radical and explore energetic
feasiblity of various routes to carbon-nitroen bond breaking at carbon 2. We have also used EPR to examine the structure of two radicals formed in the oxidative decarboxylation of pyruvate by the thiamin pyrophosphate and iron-sulfur enzyme, pyruvate ferredoxin oxidoreductase (PFOR). We have used the EPR spectroscopic results as a basis for a new ,stepwise , oxidation sequence for PFOR.
PARTICIPANTS: Participants: Russell Poyner, Associate Scientist, Gunes Bender, Research Assistant, Ann Menefee, Research Assistant and Kenneth C. Johnson, part time instrumental specialist. Research training: Ann Menefee and Gunes Bender are both Graduate students receiving research training.
TARGET AUDIENCES: Target audiences: biochemical, biophysical, and chemical scientific research communities with the intent of increasing knowledge.
Impacts
Radicals are important intermediates in many enzymatic reactions as well as potentially harmful by-products of metabolism. These species are typically transient and uniquely suited for spectroscopic investigation. Our methods are revealing the structures of these naturally occurring species and the steps in their generation and propagation. These results are useful in understanding the biochemistry and biology of free radicals.
Publications
- Carmieli, R., Larsen, T. M., Reed, G. H., Zein, S., Neese, F. and Goldfarb, D. The Catalytic Mn2+ Sites in the Enolase-Inhibitor Complex: Crystallography, Single-Crystal EPR and DFT Calculations. J. Am. Chem. Soc. 129, 4240-4252 (2007).
- Schwartz, P. A., Lobrutto, R., Reed, G. H. and Frey, P. A. Probing Interactions from Solvent-Exchangeable Protons and Monovalent Cations with the 1,2-Propanediol-1-yl Radical Intermediate in the Reaction of Dioldehydrase. Protein Sci. 16, 1156-1164 (2007).
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Progress 01/01/06 to 12/31/06
Outputs
The electron paramagnetic resonance (EPR) spectrum of an intermediate freeze trapped during the steady state of the reaction catalyzed by the adenosylcobalamin (AdoCbl)-dependent enzyme, methylmalonyl-CoA mutase, has been studied. The EPR spectrum is that of a hybrid triplet spin system created as a result of strong electron-electron spin coupling between an organic radical and the low-spin Co2+ in cob(II)alamin. The spectrum was analyzed by simulation to obtain the zero-field splitting (ZFS) parameters and Euler angles relating the radical-to-cobalt inter-spin vector to the g axis system of the low-spin Co2+. Labeling of the substrate with 13C and 2H was used to probe the identity of the organic radical partner in the triplet spin system. The patterns of inhomogeneous broadening in the EPR signals produced by [2-13C]-methylmalonyl-CoA and [2-13C]-methylmalonyl-CoA, as well as line narrowing resulting from deuterium substitution in the substrate, were consistent with
those expected for a succinyl-CoA radical wherein the unpaired electron was centered on the carbon to the free carboxyate group of the rearranged radical. The inter-spin distance and the Euler angles were used to position this product radical into the active site of the enzyme.
Impacts
Radicals are important intermediates in many enzymatic reactions as well as potentially harmful by-products of metabolism. These species are typically transient and uniquely suited for spectroscopic investigation. Our methods are revealing the structures of these naturally occurring species and the steps in their generation and propagation. These results are useful in understanding the biochemistry and biology of free radicals.
Publications
- Sims, P. A., Menefee, A. L., Larsen, T. M., Mansoorabadi, S. O., and Reed, G. H. Structure and Catalytic Properties of an Engineered Heterodimer of Enolase Composed of One Active and One Inactive Subunit, J. Mol. Biol. 355, 422-431 (2006).
- Astashkin, A. V., Seravalli, J. Mansoorabadi, S. O., Reed, G. H., and Ragsdale, S. W. Pulsed Electron Paramagnetic Resonance Experiments Identify the Paramagnetic Intermediates in the Pyruvate Ferredoxin Oxidoreductase Catalytic Cycle, J. Am. Chem. Soc. 128, 3888-3889 (2006).
- Frey, P. A., Hegeman, A. D., and Reed, G. H., Free Radical Mechanisms in Enzymology, Chem. Rev. 106, 3302-3316 (2006).
- Poyner, R. P., Anderson, M. A., Bandarian, V., Cleland, W. W., and Reed, G. H., Probing Nitrogen Sensitive Steps in the Free Radical Mediated Deamination of Amino Alcohols by Ethanolamine Ammonia-Lyase, J. Am. Chem. Soc. 128, 7120-7121 (2006).
- Mansoorabadi, S. O., Seravalli, J., Furdui, C., Krymov, V., Gerfen, G. J., Begley, T. P., Melnick, J., Ragsdale, S. W. and Reed, G. H., EPR Spectroscopic and Computational Characterization of the Hydroxyethylidene-Thiamine Pyrophosphate Radical Intermediate of Pyruvate:Ferredoxin Oxidoreductase, Biochemistry 45, 7122-7131 (2006).
- Yoon, M., Patwardhan, A., Qiao, C., Mansoorabadi, S. O., Menefee, A. L., Reed, G. H., and Marsh, E. N. G., The Reaction of Adenosylcobalamin-Dependent Glutamate Mutase with 2-Thiolglutarate, Biochemistry 45, 11650-11657 (2006).
- Beshad, E., Ruzicka, F. J., Mansoorabadi, S. O., Chen, D., Reed, G. H., and Frey, P. A., Enantiomeric free radicals and enzymatic control of stereochemistry in a radical mechanism: the case of lysine 2,3-aminomutases, Biochemistry 45, 12639-12646 (2006).
- Mansoorabadi, S. O., Magnusson, O. T., Poyner, R. R., Frey, P. A., and Reed, G. H., Analysis of the Cob(II)alamin 5-Deoxy-3,4-Anhydroadenosyl Radical Triplet Spin System in the Active Site of Diol Dehydrase Biochemistry 45, 14362-14370 (2006).
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Progress 01/01/05 to 12/31/05
Outputs
The electron paramagnetic resonance (EPR) spectrum of an intermediate freeze trapped during the steady state of the reaction catalyzed by the adenosylcobalamin (AdoCbl)-dependent enzyme, methylmalonyl-CoA mutase, has been studied. The EPR spectrum is that of a hybrid triplet spin system created as a result of strong electron-electron spin coupling between an organic radical and the low-spin Co2+ in cob(II)alamin. The spectrum was analyzed by simulation to obtain the zero-field splitting (ZFS) parameters and Euler angles relating the radical-to-cobalt inter-spin vector to the g axis system of the low-spin Co2+. Labeling of the substrate with 13C and 2H was used to probe the identity of the organic radical partner in the triplet spin system. The patterns of inhomogeneous broadening in the EPR signals produced by [2-13C]-methylmalonyl-CoA and [2-13C]-methylmalonyl-CoA, as well as line narrowing resulting from deuterium substitution in the substrate, were consistent with
those expected for a succinyl-CoA radical wherein the unpaired electron was centered on the carbon to the free carboxyate group of the rearranged radical. The inter-spin distance and the Euler angles were used to position this product radical into the active site of the enzyme.
Impacts
Radicals are important intermediates in many enzymatic reactions as well as potentially harmful by-products of metabolism. These species are typically transient and uniquely suited for spectroscopic investigation. Our methods are revealing the structures of these naturally occurring species and the steps in their generation and propagation. These results are useful in understanding the biochemistry and biology of free radicals.
Publications
- Detecting attitudinal changes about death and dying as a result of end-of-life care curricula for medical undergraduates. J Palliat Med. 2005 Oct;8(5):975-86. PMID: 16238510 [PubMed - indexed for MEDLINE]
- Magnevad status of design improvements human blood results and preliminary sheep trial. Artif Organs. 2005 Oct;29(10):855-7. PMID: 16185350 [PubMed - indexed for MEDLINE]
- Dengue Virus (DV) enhancing antibody activity in preillness plasma does not predict subsequent disease severity or viremia in secondary DV infection.J Infect Dis. 2005 Aug 1;192(3):510-9. Epub 2005 Jul 5. Erratum in: J Infect Dis. 2005 Nov 15;192(10):1863. PMID: 15995967 [PubMed - indexed for MEDLINE]
- Organizational systems to support publicly funded tobacco treatment services.Am J Prev Med. 2005 May;28(4):338-45.PMID: 15831338 [PubMed - indexed for MEDLINE]
- Effect of cardiovascular comorbidities and concomitant aspirin use on selection of cyclooxygenase inhibitor among rheumatologists.Arthritis Rheum. 2005 Feb 15;53(1):12-7. PMID: 15696570 [PubMed - indexed for MEDLINE]
- Evaluation of the Missoula-VITAS Quality of Life Index--revised: research tool or clinical tool? J Palliat Med. 2005 Feb;8(1):121-35. PMID: 15662181 [PubMed - indexed for MEDLINE]
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Progress 01/01/04 to 12/31/04
Outputs
The electron paramagnetic resonance (EPR) spectrum of an intermediate freeze trapped during the steady state of the reaction catalyzed by the adenosylcobalamin (AdoCbl)-dependent enzyme, methylmalonyl-CoA mutase, has been studied. The EPR spectrum is that of a hybrid triplet spin system created as a result of strong electron-electron spin coupling between an organic radical and the low-spin Co2+ in cob(II)alamin. The spectrum was analyzed by simulation to obtain the zero-field splitting (ZFS) parameters and Euler angles relating the radical-to-cobalt inter-spin vector to the g axis system of the low-spin Co2+. Labeling of the substrate with 13C and 2H was used to probe the identity of the organic radical partner in the triplet spin system. The patterns of inhomogeneous broadening in the EPR signals produced by [2-13C]-methylmalonyl-CoA and [2-13C]-methylmalonyl-CoA, as well as line narrowing resulting from deuterium substitution in the substrate, were consistent with
those expected for a succinyl-CoA radical wherein the unpaired electron was centered on the carbon to the free carboxyate group of the rearranged radical. The inter-spin distance and the Euler angles were used to position this product radical into the active site of the enzyme.
Impacts
Radicals are important intermediates in many enzymatic reactions as well as potentially harmful by-products of metabolism. These species are typically transient and uniquely suited for spectroscopic investigation. Our methods are revealing the structures of these naturally occurring species and the steps in their generation and propagation. These results are useful in understanding the biochemistry and biology of free radicals.
Publications
- Reed, G. H. Radical Mechanisms in Adenosylcobalamin-Dependent Enzymes. Current Opinion in Chemical Biology 8, 477-483 (2004).
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Progress 01/01/03 to 12/31/03
Outputs
Mechanistic and structural studies on radical intermediates in the adenosylcobalamin-dependent enzyme, ethanolamine ammonia-lyase, have been initiated. Adenosylcobalamin (AdoCbl)-dependent enzymes catalyze free radical reactions in which the radical center moves from the cofactor-derived radical (5'-deoxyadenosyl radical) to a substrate-derived radical. Following rearrangement of the substrate radical to a product radical, the radical center is returned to the 5'-deoxyadenosyl moiety. The organic radical intermediates are always accompanied by (and spin coupled to) another paramagnetic species- a low spin Co(II) in the cob(II)alamin fragment of the inital cofactor. Electron paramagnetics resonance (EPR) spectroscopic measurements on intermediate states in these catalytic cycles show that the substrate or product radicals are from 6 to 11 angstroms from the cobalt in the cob(II)alamin intermediate form of the cofactor. We have carried out experiments to explore how the
'players' adjust their positions during the catalytic cycles of the enzymes. In collaboration with Prof. Perry Frey, we have examined the properties of an analog of AdoCbl 3',4'-anhydroadensylcobalamin (anAdoCbl), in which the double bond in the anhydroribosyl moiety provides stabilization of the cofactor-derived radical such that it can be observed spectroscopically. The cobalt carbon bond of the analog cleaves upon binding to ethanolamine ammonia-lyase (EAL) or to diol dehyratase (DDH) in the absence of substrates as well as in their presence. The EPR spectra show that the low spin cobalt and organic radical are in a strongly-coupled triplet spin state. Isotopic substitutions in the anhydroribosyl moiety indicate the organic radical half of the triplet as the allylically-stabilized anhydroadenosyl radical. We developed a computer program to simulate the complicated EPR patterns. The parameters arising from the simulation are then used to find the position of the radical in the
molecule-fixed axis system of the cob(II)alamin. The close spacing of the two paramagnetic species and the extensive delocalization of spin within the organic radical required a higher level of interpretation than the commonly-invoked, point dipole approximation. In the case of DDH in the presence of propanediol, a good fit was obtained with C5' of the anhydroribosyl moiety located about 3 angstroms from the cobalt. We have adapted the method of global minimization known as simulated annealing to fitting of the spectroscopic data and to finding molecular structures consistent with the EPR parameters.
Impacts
Radicals are important intermediates in many enzymatic reactions as well as potentially harmful by-products of metabolism. These species are typically transient and uniquely suited for spectroscopic investigation. Our methods are revealing the structures of these naturally occurring species and the steps in their generation and propagation. These results are useful in understanding the biochemistry and biology of free radicals.
Publications
- Mansoorabadi, S. O. and Reed, G. H. Effects of Electron Spin Delocalization and Non-collinearity of Interaction Terms in EPR Triplet Powder Patterns, in Paramagnetic Resonance of Metallobiomolecules, J. Telser, ed., ACS, Washington D. C., 82-96 (2003).
- Sims, P., Larsen, T. M., Poyner, R. R., Cleland W. W., and Reed, G. H. Reverse Protonation is the Key to General Acid-Base Catalysis in Enolase. Biochemistry 42, 8289-8306 (2003).
- Schwartz, P., LoBrutto, R., Reed, G. H., and Frey, P. A. Suicide Inactivation of Dioldehydrase by Chloroacetaldehyde: Formation of the cis-Ethansemidione Radical and the Role of a Monovalent Cation. Helv. Chim. Acta 86, 3764-3775 (2003).
- Reed, G. H. and Mansoorabadi, S. O. The Positions of Radical Intermediates in the Active Sites of Adenosylcobalamin-Dependent Enzymes. Current Opinion in Structural Biology 13, 716-721 (2003).
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