Progress 07/01/99 to 09/30/10
Outputs
OUTPUTS: During the year, manuscripts submitted for publication in the ACS journal Biochemistry were received from the publisher, the American Chemical Society, for processing in the editorial office at the University of Wisconsin-Madison. Associate Editor Perry A Frey and his assistant Marjorie Ziegler coordinated the peer review of all manuscripts. In the review process, qualified referees at the national and international levels were selected and contacted as possible reviewers of each submitted manuscript. Manuscripts were forwarded to reviewers who agreed to referee the manuscripts. Referee reports were received for each manuscript. Associate Editor Frey scrutinized each manuscript and read the referee reports and made initial decisions. The possible decisions were as follows: Accept, Accept with minor revisions, Reconsider after major revisions, Reject. Assistant Ziegler forwarded accepted manuscripts to the publisher, she returned potentially acceptable manuscripts for revision to the authors, and she informed authors of rejected manusripts of the final editorial decision. Revised manuscripts received at the editorial office from authors were dealt with as follows: Manuscripts originally judged to require minor revisions were examined by Associate Editor Frey and accepted if revisions were satisfactory. Assistant Ziegler informed authors of acceptance of their revised manuscripts. Manuscripts originally judged require major revisions were returned to the original reviewers for further consideration. Referee reports on major revisions were received and considered by Associate Editor Frey. After careful consideration, the major revised manuscripts were either accepted or rejected by Associate Editor Frey, and Assistant Ziegler informed the authors of the final decisions. PARTICIPANTS: Principal Investigator (PI): Perry Allen Frey, Associate Editor of the ACS journal Biochemistry. The PI exercised professional and administrative responsibility for all aspects of the project. Administrative Assistant: Marjorie Ziegler, Editorial Assistant to the Associate Editor of the ACS journal Biochemistry. The Administrative Assistant was responsible for communications with reviewers and authors, for maintaining files on submitted manuscripts, for ensuring security of all information relating to the review and editorial decisions on manuscripts, for maintaining records of expenditures and for claiming reimbursements from the publisher. Partner association: The American Chemical Society, publisher of the ACS journal Biochemistry Collaborators and contacts: Scientists at the national and international levels who referee manuscripts for the ACS journal Biochemistry. The database of referees consists of thousands of scientists. TARGET AUDIENCES: The target audiences for the efforts of this project include biochemists, molecular biologists, chemical biologists, medicinal chemists, microbiologists, bioorganic chemists, biophysical chemists, bioinorganic chemists, nucleic acid chemists, carbohydrate chemists, nutritional scientists, physiological chemists, and medical scientists worldwide. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Manuscripts that were accepted for publication in the ACS journal Biochemistry were published in the printed and online versions of the journal. These manuscripts reported original biochemical research on biological mechanisms in terms of the structures and functions of biological molecules. The biological mechanisms reported in the ACS journal Biochemistry include the mechanisms of enzymatic reactions, mechanisms of protein chemistry, carbohydrate processing,mechanisms of hormone and vitamin action, mechanisms of nucleic acid biosynthesis and repair, mechanisms of protein biosynthesis, mechanisms of gene expression, structures and functions of membranes, mechanisms of blood coagulation, mechanisms of hormone biosynthesis, mechanisms of biological signaling, and many other related biological mechanisms. Rejected manuscripts did not elicit any outcome or impact as a result of the editorial processing at the editorial office at the University of Wisconsin-Madison. Some of these manuscripts might later be published in other journals but not with the knowledge of participation personnel associated with this project.
Publications
- No publications emanated from the editorial office at the University of Wisconsin-Madison in 2010
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: This project administers the Editorial Office at the University of Wisconsin-Madison for the American Chemical Society journal BIOCHEMISTRY. The outputs in this project include the reception of manuscripts under consideration for publication in the aforementioned journal, the maintenance of manuscript files, the selection of appropriate reviewers for each manuscript, the establishment of contacts with the potential reviewers, the final assignment of reviewers, the reception of reviewer reports for each manuscript under consideration, the consideration of the recommendations of reviewers and determination of the propriety of each review for each manuscript, the notification of a preliminary decision to the communication author of each manuscript. Preliminary decisions include the following: accept, request revisions, major revisions and re-review, or reject. Revised manuscripts are received and final decisions reached, often after consulting the reviewers or in consultation with the Editorial Advisory Board of the journal. Final decisions are forward to the communicating author, and accepted manuscripts are forwarded to the American Chemical Society for publication. PARTICIPANTS: Individuals: Perry A. Frey,Principal Investigator; Patricia Newel and Marjorie Ziegler, Editorial Assistants. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Approximately sixty percent of manuscripts received are eventually published, generally after either minor or extensive revisions. Approximately forty percent of manuscripts received are rejected, either because the subject matter lies outside the scope of the publishing policy of the journal or because the quality of the reported research does not meet the standards of the journal
Publications
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: A triplet spin system (S=1) is detected by low-temperature electron paramagnetic resonance (EPR) spectroscopy in samples of diol dehydrase and the functional AdoCbl-analogue 5'-deoxy-3',4'-anhydroadenosylcobalamin (anAdoCbl). Different spectra are observed in the presence and absence of the substrate (R,S)-1,2-propanediol. In both cases, the spectra include a prominent half-field transition (DeltaM(S) = 2) that is a hallmark of strongly coupled triplet spin systems. The appearance of Cobalt-59 hyperfine splitting in the EPR signals and the positions (g values) of the signals in the spectra show that half of the triplet spin is contributed by the low-spin Co(2+) of cob(II)alamin. Line width effects from isotopic labeling (C-13 and deuterium) in the 5'-deoxy-3',4'-anhydroribosyl ring demonstrate that the other half of the spin triplet is from an allylic 5'-deoxy-3',4'-anhydroadenosyl (anhydroadenosyl) radical. The zero-field splitting (ZFS) tensors describing the magnetic dipole-dipole interactions of the component spins of the triplets have rhombic symmetry because of electron spin delocalization within the organic radical component and the proximity of the radical to the low-spin Co(2+). The dipole-dipole interaction was modeled as a summation of point-dipole interactions involving the spin-bearing orbitals of the anhydroadenosyl radical and cob(II)alamin. Geometries which are consistent with the ZFS tensors in the presence and absence of the substrate position the 5'-carbon of the anhydroadenosyl radical 3.5 and 4.1 A from Co2+, respectively. Homolytic cleavage of the cobalt-carbon bond of the analogue in the absence of the substrate indicates that, in diol dehydrase, binding of the coenzyme to the protein weakens the bond prior to binding of the substrate. PARTICIPANTS: Ab Arabshahi and Perry Frey TARGET AUDIENCES: The target audiences would be the biochemical, biophysical, and chemical scientific research communities with the intent of increasing and/or disseminating scientific knowledge. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Two impacts of the research are noteworthy. Prior to this research, no direct evidence implicated a 5-deoxyadenosyl radical in any AdoCbl-dependent enzymatic reaction. This research directly implicated such an intermediate and strengthened earlier postulates based on indirect evidence. Second, it had been thought that the binding of the substrate for diol dehydrase provided the energy required to cleave the cobalt-carbon bond and generate the 5-deoxyadenosyl radical. This research showed that sodium binding and not substrate binding energizes this process.
Publications
- Frey P.A., Hegeman, A.D., and Ruzicka, F.J. The radical SAM superfamily. Crit. Revs. Biochem. Mol. Biol. 43, 63-88. (2007)
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: The energetics of the reductive cleavage of S-adenosylmethionine (SAM) is an outstanding question in radical SAM enzymes. The energetics is here reported for lysine 2,3-aminomutase (LAM). The reduction potential of the [4Fe-4S]2+/1+ cluster in LAM is -0.43 V with SAM bound to the iron-sulfur cluster, 1.4 V higher than the reported value for trialkylsulfonium ions in solution. The midpoint reduction potential upon binding lysine has been estimated to be -0.6 V from the values of midpoint potentials measured with SAM bound to the cluster and alanine in place of lysine, with S-adenosylhomocysteine (SAH) bound to the cluster in the presence of lysine, and with SAH bound to the cluster in the presence of alanine or of alanine and ethylamine in place of lysine. The reduction potential for SAM has been estimated to be -0.99 V from the measured value for S-3',4'-anhydroadenosylmethionine. The reduction potential for the [4Fe-4S] cluster is lowered 0.17 V by the binding of lysine to
LAM, and the binding of SAM to the [4Fe-4S] cluster in LAM elevates its reduction potential by 0.81 V. Thus, the binding of L-lysine to LAM contributes 4 kcal/mol, and the binding of SAM to the [4Fe-4S] cluster in LAM contributes 19 kcal/mol toward lowering the barrier for reductive cleavage of SAM from 32 kcal/mol in solution to 9 kcal/mol at the active site. Reaction of adenosylcobalamin-dependent dioldehydrase (DDH) with 1,2-propanediol gives rise to a radical intermediate observable by EPR spectroscopy. This reaction requires a monovalent cation such as potassium ion. The radical signal arises from the formation of a radical pair comprised of the Co(II) of cob(II)alamin and a substrate-related radical generated upon hydrogen abstraction by the 5'-deoxyadenosyl radical. The high-field asymmetric doublet arising from the organic radical has allowed investigation of its composition and environment through the use of EPR spectroscopic techniques, indicating that the unpaired electron
of the steady-state radical couples to a proton on the C(1) hydroxyl group. Other spectroscopic experiments using EPR, ENDOR, and ESEEM spectroscopy did not implicate a direct coordination of the activating cation and the substrate derived radical intermediate. The complex of dioldehydrase with adenosylcobalamin (coenzyme B12) and potassium ion reacts with molecular oxygen in the absence of a substrate to oxidize coenzyme and inactivate the complex. In this article, high performance liquid chromatography and mass spectral analysis identify the nucleoside product as 5'-peroxyadenosine, the same as the nucleoside product in aerobic nonenzymatic photolytic cleavage of adenosylcobalamin. The oxygen inactivation of the enzyme-coenzyme complex shows an absolute requirement for the same monocations required in catalysis by dioldehydrase. The results indicate that dioldehydrase likely harvests the binding energy of the activating monocation to stimulate the homolytic cleavage of the Co-C5'
bond in coenzyme B12.
PARTICIPANTS: Perry Frey, George Reed, Russell LoBrutto, Phillip Schwartz, and Susan Wang The trainees were Phillip Schwartz and Susan Wang.
TARGET AUDIENCES: Biochemical, biophysical and chemical scientific research communities with the intent of increasing knowledge.
Impacts
The binding interactions at the active site of lysine 2,3-aminomutase (LAM) with lysine and S-adenosylmethinone (SAM) lead to a decrease in the energy barrier for one-electron reductive cleavage of SAM from 1.4 Volts in solution to 0.4 Volt at the active site of LAM. A similar phenomenon must occur at the active sites of all radical SAM enzymes. Monovalent cations activate adenosylcobalamin (coenzyme-B12)-dependent dioldehydrase (DDH). Magnetic resonance spectroscopy does not reveal any interactions of the monovalent cations with the free radical intermediates in the catalytic mechanism. Chemical evidence proves that the cobalt-carbon bond in coenzyme-B12 undergoes homolytic cleavage at the active site of DDH. Further chemical studies prove that homolytic cleavage of coenzyme-B12 at DDH requires the presence of a monovalent cation. The presence of a substrate in the absence of a monovalent cation does not facilitate homolytic cleavage of the coenzyme-B12 cofactor.
Binding energy between DDH and the monovalent cation must energized the homolytic cleavage of coenzyme-B12 in the catalytic process.
Publications
- Wong, S.C. and Frey, P.A. 2007. Binding energy in teh one-electron reductive cleavage of S-adenosylmethionine in lysine 2,3-aminomutase, a radical SAM enzyme. Biochemistry 46:12889-12895.
- Schwartz, P.A. LoBrutto, R., Reed, G.H., and Frey, P.A. 2007. Probing interactions from solvent-exchangeable protons and monvalent cations with the 1,2-propanediol-1-yl radical intermediate in the reaction of dioldehydrase. Protein Sci. 16, 1157-1164.
- Schwartz, P.A. and Frey, P.A. 2007. Dioldehydrase: an essential role for potassium ion in the homolytic cleavage of the cobalt-carbon bond in adenosylcobalamin. Biochemistry 46, 7293-7301.
- Schwartz, P.A. and Frey, P.A. 2007. 5'-Peroxyadenosine and 5'-peroxyadenosylcobalamin as intermediates in the aerobic photolysis of adenosylcobalamin. Biochemistry 46, 7284-7292.
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Progress 01/01/06 to 12/31/06
Outputs
Lysine 2,3-aminomutase (LAM) from Clostridium subterminale SB4 catalyzes the interconversion of (S)-lysine and (S)-beta-lysine by a radical mechanism involving adenosylmethionine (SAM), a [4Fe-4S] cluster, and pyridoxal 5'-phosphate (PLP). The enzyme contains conserved acidic residues and a cysteine- and arginine-rich motif, which binds iron and sulfide. The activity and iron, sulfide, and PLP analysis of site-specific mutations indicate two classes of conserved residues. Mutations of R134, D293, or D330 abolish all activity. Mutations of R130, R135, or R136 or of E6, D165, E236, or D172 dramatically decrease iron and sulfide content. Mutation of D96 decreases iron and sulfide content. R130 or E172 muations display no activity, whereas mutations at other positions display low activities. The product of yjeK in Escherichia coli is a LAM and catalyzes the isomerization of (S)- but not (R)-alpha-lysine. The turnover number for LAM from E. coli is 5.0 min-1, 0.1% of the
value for clostridial LAM. The product of the E. coli enzyme is (R)-beta-lysine, the enantiomer of the clostridial product. Beta-lysine-related radicals are observed in the reactions of both enzymes by EPR) The radical in clostridial LAM has the (S)-configuration, whereas in E. coli LAM it is the (R)-configuration. A triplet spin system (S=1) is detected by low-temperature electron paramagnetic resonance (EPR) spectroscopy in diol dehydrase and 5'-deoxy-3',4'-anhydroadenosylcobalamin (anAdoCbl). Different spectra are observed in the presence and absence of the substrate 1,2-propanediol. Line width effects from isotopic labeling in the 5'-deoxy-3',4'-anhydroribosyl ring demonstrate that half of the spin triplet is from an allylic 5'-deoxy-3',4'-anhydroadenosyl (anhydroadenosyl) radical. Homolytic cleavage of the cobalt-carbon indicates that binding of the coenzyme to the protein weakens the bond. A gene eam in Clostridium difficile encodes a protein that is homologous to LAM in other
species, but it does not have the lysyl-binding residues D293 and D330 in LAM. Instead,the C. difficile protein has Lys and Asn, respectively. The C. difficile gene has been cloned into an E. coli expression vector, expressed in E. coli, and the protein purified and characterized. The recombinant protein displays excellent activity as a glutamate 2,3-aminomutase and no activity toward L-lysine. The pKa values of L-_eta-lysine and _eta-glutamate have been titrimetrically measured and are: L-beta-lysine: pK-1 = 3.25, pK-2 = 9.30, and pK-3 = 10.5. For _eta-glutamate: pK-1 = 3.13, pK-2 = 3.73, and pK-3 = 10.1. The equilibrium constants for reactions of 2,3-aminomutases favor the _eta-isomers. The pH- and temperature dependencies of Keq for LAM have been measured. The Keq is independent of pH between pH 6 and pH 11. The K-eq is temperature-dependent and ranges from 10.9 at 4 deg to 6.8 at 65 deg. The linear van't Hoff plot shows DH = -1.4 kcal mol-1 and DS = -0.25 cal deg-1 mol-1.
Exothermicity is attributed to the greater strength of the bond carbon-nitrogen bond in L-beta-lysine than in L-lysine, and this should hold for other amino acids.
Impacts
The structure of lysine 2,3-aminomutase and the preliminary midpoint reduction potentials set the stage for discovering how electron transfer energizes the reversible cleavage of S-adenosylmethionine and initiates radical mechanisms in Radical SAM enzymes.
Publications
- S-Adenosylmethionine as an oxidant: The radical SAM superfamily. Wang, S. and Frey, P.A. (2007) Trends in Biochemical Sciences, In press
- How an enzyme tames reactive intermediates: positioning of the active-site components of lysine 2,3-aminomutase during enzymatic turnover as determined by ENDOR spectroscopy. Lees, N.S., Chen, D., Walsby, C.J., Behshad, E., Frey, P.A., and Hoffman, B.M. (2006). J. Am. Chem. Soc. 128, 10145-10154.
- Enantiomeric free radicals and enzymatic control of stereochemistry in a radical mechanism: the case of lysine 2,3-aminomutases. Behshad, E., Ruzicka, F..J., Mansoorabadi, S., Reed, G.H., and Frey, P.A. (2006) Biochemistry 45, 12639-12646.
- Identification of structural and catalytic classes of highly conserved amino acid residues in lysine 2,3-aminomutase. Chen, D., Frey, P.A., Lepore, B.W., Ringe, D., and Ruzicka, F.J. (2006) Biochemistry 45, 12647-12653.
- Analysis of the cob(II)alamin-5-deoxy-3,4-anhydroadenosyl radical triplet spin system in the active site of diol dehydrase. Mansoorabadi, S.O., Magnusson, O.Th., Poyner, r.R., Frey, P.A., and Reed, G.H. (2006) Biochemistry 45, 4362-4370.
- Snapshots of three radical intermediates at the active site of pyruvate oxidase. Frey, P.A. (2006) Nat. Chem. Biol. 2, 294-295.
- Glutamate 2,3-aminomutase: A new member of the radical SAM superfamily of enzymes. Ruzicka, F.J. and Frey, P.A. (2007) Biochim. Biophys. Acta, In press.
- Basis for the equilibrium constant in the interconversion of L-lysine and L-b-lysine by lysine 2,3-aminomutase. (2007) Biochim. Biophys. Acta, In press.
- Isotope effects in the characterization of low barrier hydrogen bonds. Frey, P A. (2006) In: Isotope Effects in Chemistry and Biology (A. Kohen and H.-H. Limbach, eds.) CRC Press, Taylor and Francis Group, Boca Raton, FL, Ch. 40, pp. 975-993.
- Free radical mechanisms in enzymology. Frey, P.A., Hegeman, A.D., and Reed, G.H. (2006) Chem. Rev. 106, 3302-3316.
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Progress 02/03/05 to 02/02/06
Outputs
Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of L-lysine and L-b-lysine by a free radical mechanism. The 5-deoxyadenosyl radical derived from the reductive cleavage of S-adenosyl-L-methionine (SAM) initiates substrate-radical formation. The [4Fe-4S]1+ cluster in LAM is the one-electron source in the reductive cleavage of SAM, which is directly ligated to the unique iorn site in the cluster. We here report the midpoint reduction potentials of the [4Fe-4S]2+/1+ couple in the presence of SAM, S-adenosyl-L-homocysteine (SAH), or 5-[N-[(3S)-3-amino-carboxypropyl]-N-methylamino]-5-deoxyadenosine (azaSAM) as measured by spectroelectrochemistry. The reduction potentials are -430 mV in the presence of SAM, -460 mV in the presence of SAH, and -497 mV in the presence of azaSAM. In the absence of SAM or an analog and the presence of dithiothreitol, dihydrolipoate, or cysteine as ligands to the unique iron, the midpoint potentials are -479 mV, -516 mV, or -484 mV,
respectively. LAM is a member of the Radical SAM superfamily of enzymes, in which the CxxxCxxC modif donates three thiolate ligands to iron in the [4Fe-4S] cluster and SAM donates the amino and carboxylate groups of the methionyl moiety as ligands to the fourth iron. The results show the reduction potentials in the midrange for ferredoxin-like [4Fe-4S] clusters. They show that SAM elevates the reduction potential by 86 mV relative to dihydrolipoate as the cluster ligand. This difference accounts for the SAM-dependent reduction of the [4Fe-4S]2+ cluster by dithionite reported earlier. Analogs of SAM have a diminished capacity to raise the potential. The X-ray crystal structure of the pyridoxal-5-phosphate (PLP), S-adenosyl-L-methionine (SAM), and [4Fe-4S]-dependent lysine-2,3-aminomutase (LAM) of Clostridium subterminale has been solved to 2.1 Angstrom resolution by single wavelength anomalous dispersion methods on a L- selenomethionine substituted complex of LAM with [4Fe-04S]2+, PLP,
SAM, and L-lysine, a very close analog of the active Michaelis complex. The unit cell contains a dimer of hydrogen-bonded, domain swapped dimers, the subunits of which adopt a fold that contains all three cofactors in a central channel defined by six beta/alpha structural units. Zinc coordination links the domain-swapped dimers. In each subunit the solvent-face of the channel is occluded by an N-terminal helical domain, with the opposite end of the channel packed against the domain swapped subunit. Hydrogen bonded ionic contacts hold the external aldimine of PLP and L-lysine in position for abstraction of the 3-pro-R hydrogen of lysine by C5 of SAM. The structure of the SAM/[4Fe-4S] complex confirms and extends conclusions from spectroscopic studies of LAM and shows selenium in Se-adenosyl-L-selenomethionine poised to ligate the unique iron in the [4Fe-4S] cluster upon electron transfer and radical formation.
Impacts
The structure of lysine 2,3-aminomutase and the preliminary midpoint reduction potentials set the stage for discovering how electron transfer energizes the reversible cleavage of S-adenosylmethionine and initiates radical mechanisms in Radical SAM enzymes.
Publications
- Hinckley GT, Frey PA. (2006) An adaptable spectroelectrochemical titrator: The midpoint reduction potential of the iron-sulfur center in lysine 2,3-aminomutase. Anal Biochem. 349,103-11.
- Lepore BW, Ruzicka FJ, Frey PA, Ringe D. (2005) The x-ray crystal structure of lysine-2,3-aminomutase from Clostridium subterminale. Proc Natl Acad Sci U S A 102, 13819-24.
- Hinckley GT, Frey PA (2006) Cofactor-Dependence in Reduction Potentials for [4Fe-4S]2+/1+ in Lysine 2,3-Aminomutase. Biochemistry, In press.
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Progress 01/01/04 to 12/31/04
Outputs
Lysine 2,3-aminomutase (LAM). LAM catalyzes the interconversion of lysine and beta-lysine by a radical mechanism initiated by scission of the carbon-sulfur bond in S-adenosylmethionine (SAM) to methionine and the adenosyl radical. The electron required arises from an iron-sulfur cluster. Selenium x-ray absorption spectroscopy in the reaction with (SeSAM) shows that an inner sphere electron transfer culminates in direct ligation of selenomethionine to iron upon cleavage of SeSAM. We show through electron nuclear double resonance (ENDOR) spectroscopy that SAM binds to LAM by direct ligation to iron in the cluster through the carboxyl and amine groups of methionine. Based on the ENDOR and XAS, we postulate a mechanism for the cleavage of SAM through multiple ligation with the cluster. Lysine 5,6-aminomutase (5,6-LAM). 5,6-LAM catalyzes the transformations of lysine into 2,5-diaminohexanoate (2,5-DAH) and of beta-lysine into 3,5-diaminohexanoate (3,5-DAH). The activity
depends on pyridoxal-5-phosphate (PLP) and coenzyme B12. The postulated mechanism requires twelve steps, two involving hydrogen transfer. The deuterium kinetic isotope effects on V and V/M have been found to be 10.4 and 8.3 respectively, for reaction of lysine. Neither cob(II)alamin nor a free radical can be detected in the steady state. Therefore, hydrogen abstraction from carbon-5 of the substrate side chain is rate limiting. 4-Oxalysine is an alternative substrate and reacts irreversibly because the product breaks down into ammonia, acetaldehyde, and serine. Km for 4-oxalysine is lower than for lysine, and Vm is also lower. As measured by V/K, 5,6-LAM uses 4-oxalysine as efficiently as the best substrates. 4-Oxalysine induces the same suicide inactivation by electron transfer as do the biological substrates. Dioldehydrase. Dioldehydrase catalyzes the Vitamin B12-depenent dehydration of 1,2-propanediol to propionaldehyde. The reaction proceeds by a radical mechanism initiated by
scission of the cobalt-carbon bond to form the adenosyl radical. Dioldehydrase undergoes suicide inactivation by chloroacetaldehyde, which becomes the cis-ethanesemidione radical. Chloroacetaldehyde reacts in the reverse catalytic process to a rearranged radical that eliminates HCl and leads to cis-ethanesemidione. Potassium or thallous ions, are required for activity and for suicide inactivation. Electron spin resonance (ESR) experiments show that the magnetic nucleus of thallous ion does not interact with the cis-ethanesemidione radical. Pulsed ESR experiments implicate an amino acid, possibly of His143, interacting with the radical. Ribonucleotide reductase (RTPR). The Vitamin B12-dependent RTPR catalyzes the reduction of ribonucleoside triphosphates to deoxyribonucleoside triphosphates. A thiyl radical on Cys 408 arises from coenzyme B12 and is proposed to mediate nucleotide reduction. A stereochemical test of the mechanism of the cobalt-carbon bond cleavage of proved that the C5
of adenosyl in coenzyme B12 was epimerized by the wild-type and C408A-RTPR. The results prove that, contrary to earlier claims, the SH group of Cys408 does not participate in the RTPR-mediated carbon-cobalt bond homolysis.
Impacts
The functions of the enzymes under investigation depend on Vitamin B12, Vitamin B6, S-adenosylmethionine, and iron, all of which are nutritionally active in the human diet. The functions of these molecules are under investigation. The enzyme lysine 2,3-aminomutase produces molecules that may be used in the synthesis of pharmaceutical agents.
Publications
- Suicide Inactivation of Dioldehydrase by Chloroacetaldehyde: Formation of the cis-Ethanesemidione Radical and the Role of a Monovalent Cation. Schwartz, P., LoBrutto, R., Reed, G.H. and Frey, P.A. (2004) Helv. Chim. Acta 86, 3764-3775.
- A Locking Mechanism Preventing Radical Damage in the Absence of Substrate, as Revealed by the X-ray Structure of Lysine 5,6-Aminomutase. Berkovitch, F., Behshad, E, Tang, K.-H., Enns, E.A., Frey, P.A, and Drennan, C.L. (2004) Proc. Nat'l. Acad. Sci. 101, 15870-15875.
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Progress 01/01/03 to 12/31/03
Outputs
Lysine 2,3-aminomutase (LAM) catalyzes interconversion of L-lysine and L-Beta-lysine by a radical mechanism initiated by scission of C5'-S bond in S-adenosylmethionine (SAM) to methionine and 5'-deoxyadenosyl radical. The electron required in cleavage of SAM arises from [4Fe-4S]+ cluster associated with this enzyme. Little is known of the mechanism by which the electron is inserted into SAM to effect cleavage. Selenium x-ray absorption spectroscopy in reaction of Se-adenosyl-L-selenomethionine (SeSAM) in place of SAM shows that electron transfer from [4Fe-4S] center occurs by an inner sphere mechanism culminating in direct ligation of selenomethionine to iron upon cleavage of SeSAM. We show through electron nuclear double resonance (ENDOR) spectroscopy that SAM binds to LAM by direct ligation to the iron in [4FE-4S] center through carboxyl and amine groups of its L-methionine. Based on ENDOR and XAS, we postulate a mechanism for cleavage of SAM through multiple
ligation with the [4Fe-4S] center. Lysine 5,6-aminomutase (5,6-LAM) catalyzes essentially isoenergetic transformations of D-lysine in 2,5-diaminohexanoate (2,5-DAH) and of L-beta-lysine into 3,5-diaminohexanoate (3,5-DAH). Activity of 5,6-LAM depends on pyridoxal-5'-phosphate (PLP) and adensylcobalamin. The currently postulated mechanism requires twelve steps, two involving hydrogen transfer. Deuterium kinetic isotope effects on V and V/M have been found to be 10.4 +/- 0.3 and 8.3 +/- 1.9, respectively, for reaction of DL-lysine-3,3,4,4,5,5,6,6,-d. Isotope effects for reaction of DL-lysine-4,4,5,5,-d are 8.5 +/- 0.7 and 7.1 +/- 1.2. Neither cob(II)alamin nor a free radical can be detected in the steady state. Therefore, hydrogen abstraction from carbon-5 of the substrate side chain is rate limiting. DL-4-Oxalysine is an alternative substrate for 5,6-LAM. DL-4-Oxalysine reacts irreversibly because the product breaks down into ammonia, acetaldehyde, and DL-serine. Km for DL-4-oxalysine
is lower than for DL-lysine, and Vm for DL-4-oxalysine is lower than for DL-lysine. As measured by V/K, 5,6-LAM uses DL-4-oxalysine as efficiently as the best substrates. DL-4-Oxalysine induces the same suicide inactivation by electron transfer as do biological substrates.
Impacts
The functions of the enzymes under investigation depend on Vitamin B12, Vitamin B6, S-adenosylmethionine, and iron, all of which are nutritionally active in the human diet. The functions of these molecules are under investigation. The enzyme lysine 2,3-aminomutase produces molecules that may be used in the synthesis of pharmaceutical agents.
Publications
- Epimerization at carbon-5' of (5'R)-[5'-2H] adenosylcobalamin by ribonucleoside triphosphate reductase: Cysteine 408-independent cleavage of the Co-C5' bond. Chen, D., Abend, A., Stubbe, J., and Frey, P. A. (2003) Biochemistry 42, 4578-4584.
- Adenosyl coenzyme and pH dependence of the [4Fe-4S]2+/1+ transition in lysine 2,3-aminomutase. Hinckley, G. T., Ruzicka, F. J., Thompson, M. J., Blackburn, G. M., and Frey, P. A. (2003) Arch Biochem Biophys. 414, 34-39.
- Kinetic and biochemical analysis of the mechanism of action of lysine 5,6-aminomutase. Tang, K. H., Casarez, A. D., Wu, W., and Frey, P. A. (2003) Arch. Biochem. Biophys. 418, 49-54.
- Coordination and mechanism of reversible cleavage of S-adenosylmethionine by they [4Fe-4S] center in lysine 2,3-aminomutase. Chen, D., Walsby, C., Hoffman, B. M., and Frey, P. A. (2003) J. Am. Chem. Soc. 125, 11788-11789.
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Progress 01/01/02 to 12/31/02
Outputs
Lysine 5,6-aminomutase (5,6-LAM) catalyzes the interconversion of D-lysine with 2,5-diaminohexanoate and of L-beta-lysine with 3,5- diaminohexanoate. The coenzymes for 5,6-LAM are adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP). In the proposed chemical mechanism, AdoCbl initiates the formation of substrate-radicals, and PLP facilitates the radical rearrangement by forming an external aldimine linkage with the e-amino group of a substrate, either D-lysine or L-beta-lysine. In the resting enzyme, an internal aldimine between PLP and an essential lysine in the active site facilitates productive PLP binding and catalysis. We present here biochemical, biophysical, and site-directed mutagenesis experiments, which document the existence of an essential lysine residue in the active site of 5,6-LAM from P. gingivalis. Reduction of 5,6-LAM with NaBH4 rapidly inactivates the enzyme and shifts the electronic absorption band from 420 to 325 nm. This is characteristic
of the reduction of an aldimine linkage between the carbonyl group of PLP and the e-amino group of a lysine residue. The reduced peptide was identified by Q-TOF/MS, and further confirmed by Q-TOF/MS/MS sequencing. We show that lysine 144 in the small subunit of 5,6-LAM is the essential lysine residue. Lysine 144(beta) is separated by only eleven amino acids from histidine 133(beta), which forms a part of the 'base-off'-AdoCbl binding motif. The sequence of the novel PLP binding motif is conserved in 5,6-LAM from C. sticklandii and P. gingivalis, and it is distinct from all known PLP-binding motifs. Mutation of lysine 144(beta) to glutamine led to K144Q(beta)-5,6-LAM, which displayed no enzymatic activity and no absorption band corresponding to an internal PLP-aldimine. In summary, we introduce a novel PLP-binding motif, the first to be discovered in an AdoCbl dependent enzyme.
Impacts
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Publications
- Hydrogen-Deuterium Exchange at the 5'-Position of an Analogue of S-Adenosyl-L-methionine. Magnusson, O. Th. and Frey, P. A. (2002) Bioorganic Chemistry 30, 53-61.
- Interactions of Diol Dehydrase and 3',4'-Anhydroadenosylcobalamin: Suicide Inactivation by Electron Transfer. Magnusson, O. Th. and Frey, P. A. (2002) Biochemistry 41, 1695-1702.
- Identification of a Novel Pyridoxal 5'-Phosphate Binding Site in Adenosylcobalamin-Dependent Lysine 5,6-Aminomutase from Porphyromonas gingivalis. Tang, K.H., Harms, A., and Frey, P. A. (2002) Biochemistry 41, 8767-8776.
- Kinetic characterization of transient free radical intermediates in the reaction of lysine 2,3-aminomutase by EPR lineshape analysis. Frey, P. A., Chang, C. H., Ballinger, M. D., and Reed, G. H. (2002) Methods Enzymol. 354, 426-435.
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Progress 01/01/01 to 12/31/01
Outputs
The dTDP-glucose 4,6-dehydratase catalyzed conversion of dTDP-glucose to dTDP-4-keto-6-deoxyglucose occurs in three sequential chemical steps: dehydrogenation, dehydration, and re-reduction. The enzyme contains the tightly bound coenzyme NAD+, which mediates the dehydrogenation and re-reduction steps of the reaction mechanism. In this study, we have identified the amino acid residues participating in acid/base catalysis of the dehydration step. To accomplish this, we have analyzed wild type and variant dTDP-glucose 4,6-dehydratases in the context of two novel assays. First, we have synthesized an alternative substrate, dTDP-6-fluoro-6-deoxyglucose (dTDP-6FGlc), which undergoes fluoride ion elimination instead of dehydration, and thus does not require acid catalysis. The mechanistic consequences resulting from use of dTDP-6FGlc were analyzed during net turnover. Second, we have identified a previously uncharacterized, enzyme-catalyzed glucosyl-C5 hydrogen-solvent
exchange reaction of the product, dTDP-4-keto-6-deoxyglucose. Since base catalysis of this exchange reaction is analogous to that occurring at C5 during the dehydration step of net catalysis, analysis of variants, performance of this reaction reveals the corresponding residue. Using these unique mechanistic approaches, we have unambiguously identified the acid and base in the dehydration step as Asp135 and Glu136, respectively.
Impacts
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Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
The steady state kinetic parameters for epimerization of UDP-galactose by UDP-galactose 4-epimerase from Escherichia coli (GalE), Y149F-GalE, and S124A-GalE have been measured as a function of pH. The deuterium kinetic isotope effects for epimerization of UDP-galactose-C-d7 by these enzymes have also been measured. The results show that the activity of wild type GalE is pH-independent in the range of pH 5.5 to 9.3, and there is no significant deuterium kinetic isotope effect in the reaction of UDP-galactose-C-d7. It is concluded that the rate-limiting step for epimerization by wild type GalE is not hydride transfer and must be either a diffusional process or a conformational change. Epimerization of UDP-galactose-C-d7 by Y14F-GalE proceeds with a pH-dependent deuterium kinetic isotope effect on kcat of 2.2 plus/minus 0.4 at pH 8.3 and 1.1 plus/minus 0.5 at pH 6.2. Moreover, the plot of log kcat/Km breaks downward on the acid side with a fitted value of 7.1 for the
pKa. It is concluded that the break in the pH-rate profile arises from a change in rate limiting step from hydride transfer at low pH to a conformational change at high pH. Epimerization of UDP-galactose-C-d7 by S124A-GalE proceeds with a pH-independent deuterium kinetic isotope effect on kcat of 2.0 plus/minus 0.2 between pH 6 and pH 9. Both plots of log kcat and log kcat/Km display pH-dependence. The plot of log kcat against pH breaks downward with a pKa of 6.35 plus/minus 0.10. The plot of log kcat/Km against pH is bell-shaped, with fitted values of pKa of 6.6 plus/minus 0.09 and 9.32 plus/minus 0.21. It is concluded that hydride transfer is rate limiting, and the pKa when measured spectrophotometrically in this variant. Acid-base catalysis by Y149F-GalE is attributed to Ser 124, which is postulated to rescue catalysis of proton transfer in the absence of Tyr 149. The kinetic pKa of 7.1 for free Y149F-GalE is lower than that expected for Ser 124, as proven by the pH-dependent
kinetic isotope effect. Epimerization by the doubly mutated Y149F/S124A-GalE proceeds at value of kcat that is lower by a factor of 10 to the 7th than wild type GalE. This low rate is attributed to the synergistic actions of Tyr 149 and Ser 124 in wild type GalE. This low rate is attributed to the synergistic actions of Tyr 149 an Ser 124 in wild type GalE and to the absence of any internal catalysis of hydride transfer in doubly mutated enzyme.
Impacts
(N/A)
Publications
- No publications reported this period
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