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Query: UMLS:C0162473 (Frey)
 2,599 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The distribution of DNP groups in various tissues of guinea-pigs following intravenous injection of DNBSO3Na was investigated by an immunofluorescent method using FITC-labelled anti-DNP antibody. DNP groups were detected either in the cytoplasms of epidermal cells or on/in lymphoid tissue cells. The epidermal distribution of DNP groups was shown in the skin obtained from 5 min to 3 days after injection. There was no fundamental difference in the epidermal localization of DNP groups between at the previously tested site and at virgin site in DNCB sensitized animals. Unreacted DNBSO3Na was demonstrated in the blood plasma of DNBSO3Na injected animals. The injection of either DNP-lysine or 2,4-dinitrophenol was found incapable of inducing flare up reaction and no epidermal localization of DNP groups in the sensitized animals was observed. A possibility that intravenously injected DNBSO3Na forms a complete antigen by conjugation with epidermal proteins and sensitized cells, which have been indicated by Polak, Turk & Frey (1973) to remain at the site of old contact reaction, react with the antigen resulting in flare up reaction, is suggested.
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PMID:The distribution of antigen in flare up reaction in contact sensitivity to DNCB. 64 Jul 19

Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of L-alpha-lysine with L-beta-lysine. The purified enzyme contains iron-sulfur ([Fe-S]) clusters, pyridoxal phosphate, and Co(II) [Petrovich, R. M., Ruzicka, F. J., Reed, G. H., & Frey, P. A. (1991) J. Biol. Chem. 266, 7656-7660]. Enzymatic activity depends upon the presence and integrity of these cofactors. In addition, the enzyme is activated by S-adenosylmethionine, which participates in the transfer of a substrate hydrogen atom between carbon-3 of lysine and carbon-2 of beta-lysine [Moss, M., & Frey, P. A. (1987) J. Biol. Chem. 262, 14859-14862]. This paper describes the electron paramagnetic resonance (EPR) properties of the [Fe-S] clusters. Purified samples of the enzyme also contain low and variable levels of a stable radical. The radical spectrum is centered at g = 2.006 and is subject to inhomogeneous broadening at 10 K, with a p1/2 value of 550 +/- 100 microW. The low-temperature EPR spectrum of the [Fe-S] cluster is centered at g = 2.007 and undergoes power saturation at 10 K in a homogeneous manner, with a p1/2 of 15 +/- 2 mW. The signals are consistent with the formulation [4Fe-4S] and are adequately simulated by a rhombic spectrum, in which gxx = 2.027, gyy = 2.007, and gzz = 1.99. Treatment of the enzyme with reducing agents converts the cluster into an EPR-silent form. Oxidation of the purified enzyme by air or ferricyanide converts the [Fe-S] complex into a species with an EPR spectrum that is consistent with the formulation [3Fe-4S].(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of iron-sulfur clusters in lysine 2,3-aminomutase by electron paramagnetic resonance spectroscopy. 132 54

Electron paramagnetic resonance (EPR) spectroscopy has been used to characterize an organic radical that appears in the steady state of the reaction catalyzed by lysine 2,3-aminomutase from Clostridium SB4. Results of a previous electron paramagnetic resonance (EPR) study [Ballinger, M. D., Reed, G. H., & Frey, P. A. (1992) Biochemistry 31, 949-953] demonstrated the presence of EPR signals from an organic radical in reaction mixtures of the enzyme. The materialization of these signals depended upon the presence of the enzyme, all of its cofactors, and the substrate, lysine. Changes in the EPR spectrum in response to deuteration in the substrate implicated the carbon skeleton of lysine as host for the radical center. This radical has been further characterized by EPR measurements on samples with isotopically substituted forms of lysine and by analysis of the hyperfine splittings in resolution-enhanced spectra by computer simulations. Changes in the hyperfine splitting patterns in EPR spectra from samples with [2-2H]lysine and [2-13C]-lysine show that the paramagnetic species is a pi-radical with the unpaired spin localized primarily in a p orbital on C2 of beta-lysine. In the EPR spectrum of this radical, the alpha-proton, the beta-nitrogen, and the beta-proton are responsible for the hyperfine structure. Analysis of spectra for reactions initiated with L-lysine, [3,3,4,4,5,5,6,6-2H8]lysine, [2-2H]lysine, perdeuteriolysine, [alpha-15N]lysine, and [alpha-15N,2-2H]lysine permit a self-consistent assignment of hyperfine splittings.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Structure of a substrate radical intermediate in the reaction of lysine 2,3-aminomutase. 132 55

Structural studies carried out on a cross-linked complex between cytochrome c3 and ferredoxin I, both isolated from Desulfovibrio desulfuricans Norway, allowed the identification of the site of interaction between the two redox proteins. Staphylococcus aureus proteinase and chymotrypsin digestions led to characterization of peptides containing both cytochrome c3 and ferredoxin sequences. The cytochrome c3 sequences involved in the three isolated cross-linked peptides contained several lysine residues localized around the heme 4 crevice. This analysis stressed the peculiar role of lysines 100, 101, 103, 104 and 113, which could be considered as major cross-link sites, as opposed to the lysines 75, 79 and 82, which could be considered as minor cross-link sites. One cross-linked peptide, containing two ferredoxin sequences joined to one cytochrome c3 sequence, had been isolated, suggesting the possibility of more than one cross-link per covalent complex. All these results led to the identification of heme 4 of cytochrome c3 as the site of interaction for the ferredoxin I. This study confirms the proposal that could be deduced from the hypothetical structure of the complex built by computer graphics modelling (Cambillau, C., Frey, M., Mosse, J., Guerlesquin, F. and Bruschi, M. (1988) Proteins: struct., funct. genet. 4, 63-70).
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PMID:Identification of the site of interaction between cytochrome c3 and ferredoxin using peptide mapping of the cross-linked complex. 164 31

Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of lysine and beta-lysine by a mechanism in which four organic radicals are postulated as intermediates. One of the intermediates has been identified as the alpha-radical of beta-lysine in imine linkage to pyridoxal phosphate (PLP) [Ballinger, M. D., Frey, P. A., & Reed, G. H. (1992) Biochemistry 31, 10782-10788]. We report here the observation of another of the four putative radical intermediates in the reaction of the alternative substrate, 4-thia-L-lysine (S-2-aminoethyl-L-cysteine). 4-Thialysine is a substrate for lysine 2,3-aminomutase. The Km of 4-thialysine is similar to that for lysine, and the Vm is approximately 3% of that for lysine. Upon mixing 4-thialysine with the activated enzyme in the presence of the required cofactor S-adenosylmethionine, followed by freeze-quenching with liquid N2 in the steady state, a strong EPR signal centered at g = 2.003 is observed. This signal exhibits strong hyperfine splitting due to the presence of 13C at carbon-3 of 4-thialysine, and the EPR pattern is narrowed upon the substitution of deuterium at carbon-3. The hyperfine interactions show that the unpaired electron is centered on carbon-3 of 4-thialysine. The hyperfine pattern in the EPR spectrum is also simplified by the use of 4-thia[5,6-2H4]lysine as the substrate, showing either that the spin is partially delocalized through the sulfur intervening between carbons-3 and -5 or that the conformation is such that protons at carbon-6 are close to carbon-3.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Observation of a second substrate radical intermediate in the reaction of lysine 2,3-aminomutase: a radical centered on the beta-carbon of the alternative substrate, 4-thia-L-lysine. 765 8

Acetoacetate decarboxylase from Clostridium acetobutylicum (AAD) catalyzes the decarboxylation of acetoacetate via a Schiff base intermediate [Hamilton, G. A., & Westheimer, F. H. (1959) J. Am. Chem. Soc. 81, 6332; Fridovich, I., & Westheimer F. H. (1962) J. Am. Chem. Soc. 84, 3208]. The pKa of the active-site lysine (Lys 115) is 6.0, 4.5 pKa units less than the pKa of lysine in solution [Kokesh, F. C., & Westheimer, F. H. (1971) J. Am. Chem. Soc. 93, 7270; Frey, P. A., Kokesh, F. C., & Westheimer, F. H. (1971) J. Am. Chem. Soc. 93, 7266; Schmidt, D. E., Jr., & Westheimer, F. H. (1971) Biochemistry 10, 1249]. Westheimer and co-workers hypothesized that the pKa of Lys 115 is decreased by its spatial proximity to the epsilon-ammonium group of Lys 116. We have investigated this proposal by studying site-directed mutants of Lys 115 and Lys 116. Two substitutions for Lys 115 (K115C and K115Q) were both catalytically inactive at pH 5.95, the pH optimum of wild type AAD, demonstrating the importance of this residue in catalysis. Activity could be restored to K115C by aminoethylation with 2-bromoethyl-ammonium bromide (2-BEAB). Substitutions for Lys 116 (K116C, K116N, and K116R) had reduced but significant activities at pH 5.95. The effects of Lys 116 on the pKa of Lys 115 in the mutant AADs were evaluated following imine formation with 5-nitrosalicylaldehyde and reduction with NaBH4. Whereas the pKa of Lys 115 in K116R is similar to that observed for wild type AAD, the pKaS of Lys 115 in K116C and K116N were elevated to > 9.2. Alkylation of Cys 116 in K116C with 2-BEAB resulted in both significant activation and restoration of the pKa of Lys 115 to 5.9. These data support Westheimer's hypothesis that the pKa of the Schiff base-forming Lys 115 is decreased by its spatial proximity to the epsilon-ammonium group of Lys 116.
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PMID:Mechanism of the reaction catalyzed by acetoacetate decarboxylase. Importance of lysine 116 in determining the pKa of active-site lysine 115. 855 96

UDP-galactose 4-epimerase from Escherichia coli catalyzes the interconversion of UDP-glucose and UDP-galactose. In recent years, the enzyme has been the subject of intensive investigation due in part to its ability to facilitate nonstereospecific hydride transfer between beta-NADH and a 4-keto hexopyranose intermediate. The first molecular model of the epimerase from E. coli was solved to 2.5 A resolution with crystals grown in the presence of a substrate analogue, UDP-phenol (Bauer AJ, Rayment I, Frey PA, Holden HM, 1992, Proteins Struct Funct Genet 12:372-381). There were concerns at the time that the inhibitor did not adequately mimic the sugar moiety of a true substrate. Here we describe the high-resolution X-ray crystal structure of the ternary complex of UDP-galactose 4-epimerase with NADH and UDP-phenol. The model was refined to 1.8 A resolution with a final overall R-factor of 18.6%. This high-resolution structural analysis demonstrates that the original concerns were unfounded and that, in fact, UDP-phenol and UDP-glucose bind similarly. The carboxamide groups of the dinucleotides, in both subunits, are displaced significantly from the planes of the nicotinamide rings by hydrogen bonding interactions with Ser 124 and Tyr 149. UDP-galactose 4-epimerase belongs to a family of enzymes known as the short-chain dehydrogenases, which contain a characteristic Tyr-Lys couple thought to be important for catalysis. The epimerase/NADH/UDP-phenol model presented here represents a well-defined ternary complex for this family of proteins and, as such, provides important information regarding the possible role of the Tyr-Lys couple in the reaction mechanism.
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PMID:High-resolution X-ray structure of UDP-galactose 4-epimerase complexed with UDP-phenol. 893 Nov 34

Lysine 2,3-aminomutase catalyzes the interconversion of L-lysine and L-beta-lysine. 4-Thia-L-lysine (4-thialysine) is an alternative substrate for Lysine 2,3-aminomutase. The organic free radical that appears in the steady state of the reaction of 4-thialysine is structurally analogous to the first lysine-based radical in the chemical mechanism (Wu, W., Lieder, K. W., Reed, G. H., and Frey, P. A. (1995) Biochemistry 34, 10532-10537). 4-Thialysine is a much more potent inhibitor of the reaction of lysine than would be anticipated on the basis of the value of Km for its reaction as a substrate. 4-Thialysine is here shown to be a competitive reversible inhibitor with respect to L-lysine, displaying an inhibition constant of 0.12 +/- 0.01 mM. The value of Km for 4-thialysine is 1.4 +/- 0.1 mM, and the maximum velocity Vm = 0.19 +/-0.02 micromol min(-1) mg-1 at 37 degrees C and pH 8.0. The kinetic parameters for the reaction of lysine under the same conditions are: Km = 4.2 +/- 0.5 mM and Vm = 43 +/- 1 micromol min(-1) mg(-1). The discrepancy between Km and the apparent Ki for 4-thialysine arises from the fact that the maximal velocity for 4-thialysine is only 0.44% that for L-lysine. The electron paramagnetic resonance spectra of the organic radical generated at the active site from 4-thialysine and those generated from deuterium and 3-13C-labeled forms of 4-thialysine were analyzed by simulation. Based on the resulting hyperfine splitting constants, the conformation and distribution of the unpaired spin of the radical at the active site were evaluated.
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PMID:Inhibition of lysine 2,3-aminomutase by the alternative substrate 4-thialysine and characterization of the 4-thialysyl radical intermediate. 1137 Aug 52

An allylic analogue of the 5'-deoxyadenosyl radical has been characterized at the active site of lysine 2,3-aminomutase (LAM) by electron paramagnetic resonance (EPR) spectroscopy. The anhydroadenosyl radical, 5'-deoxy-3',4'-anhydroadenosine-5'-yl, is a surrogate of the less stable 5'-deoxyadenosyl radical, which has never been observed but has been postulated to be a radical intermediate in the catalytic cycles of a number of enzymes. An earlier communication [Magnusson, O.Th., Reed, G. H., and Frey, P. A. (1999) J. Am. Chem. Soc. 121, 9764-9765] included the initial spectroscopic identification at 77 K of the radical, which is formed upon replacement of S-adenosylmethionine by S-3',4'-anhydroadenosylmethionine as a coenzyme for LAM. The electron paramagnetic resonance spectrum of the radical changes dramatically between 77 and 4.5 K. This unusual temperature dependence is attributed to a spin-spin interaction between the radical and thermally populated, higher spin states of the [4Fe-4S]+2 center, which is diamagnetic at 4.5 K. The EPR spectra of the radical at 4.5 K have been analyzed using isotopic substitutions and simulations. Analysis of the nuclear hyperfine splitting shows that the unpaired spin is distributed equally between C5'- and C3'- as expected for an allylic radical. Hyperfine splitting from the beta-proton at C-2'(H) shows that the dihedral angle to the p(z)-orbital at C-3' is approximately 37 degrees. This conformation is in good agreement with a structural model of the radical. The rate of formation of the allylic radical shows that it is kinetically competent as an intermediate. Measurements of 2H kinetic isotope effects indicate that with lysine as the substrate, the rate-limiting steps follow initial reductive cleavage of the coenzyme analogue.
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PMID:Characterization of an allylic analogue of the 5'-deoxyadenosyl radical: an intermediate in the reaction of lysine 2,3-aminomutase. 1142 3

We report here the x-ray crystal structure of a soluble catalytically active fragment of the Escherichia coli type I signal peptidase (SPase-(Delta2-75)) in the absence of inhibitor or substrate (apoenzyme). The structure was solved by molecular replacement and refined to 2.4 A resolution in a different space group (P4(1)2(1)2) from that of the previously published acyl-enzyme inhibitor-bound structure (P2(1)2(1)2) (Paetzel, M., Dalbey, R.E., and Strynadka, N.C.J. (1998) Nature 396, 186-190). A comparison with the acyl-enzyme structure shows significant side-chain and main-chain differences in the binding site and active site regions, which result in a smaller S1 binding pocket in the apoenzyme. The apoenzyme structure is consistent with SPase utilizing an unusual oxyanion hole containing one side-chain hydroxyl hydrogen (Ser-88 OgammaH) and one main-chain amide hydrogen (Ser-90 NH). Analysis of the apoenzyme active site reveals a potential deacylating water that was displaced by the inhibitor. It has been proposed that SPase utilizes a Ser-Lys dyad mechanism in the cleavage reaction. A similar mechanism has been proposed for the LexA family of proteases. A structural comparison of SPase and members of the LexA family of proteases reveals a difference in the side-chain orientation for the general base lysine, both of which are stabilized by an adjacent hydroxyl group. To gain insight into how signal peptidase recognizes its substrates, we have modeled a signal peptide into the binding site of SPase. The model is built based on the recently solved crystal structure of the analogous enzyme LexA (Luo, Y., Pfuetzner, R. A., Mosimann, S., Paetzel, M., Frey, E. A., Cherney, M., Kim, B., Little, J. W., and Strynadka, N. C. J. (2001) Cell 106, 1-10) with its bound cleavage site region.
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PMID:Crystal structure of a bacterial signal peptidase apoenzyme: implications for signal peptide binding and the Ser-Lys dyad mechanism. 1174 64


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