Enantiomeric free radicals and enzymatic control of stereochemistry in a radical mechanism: the case of lysine 2,3-aminomutases.

The product of yjeK in Escherichia coli is a homologue of lysine 2,3-aminomutase (LAM) from Clostridium subterminale SB4, and both enzymes catalyze the isomerization of (S)- but not (R)-alpha-lysine by radical mechanisms. The turnover number for LAM from E. coli is 5.0 min(-1), 0.1% of the value for clostridial LAM. The reaction of E. coli LAM with (S)-alpha-[3,3,4,4,5,5,6,6-(2)H8]lysine proceeds with a kinetic isotope effect (kH/kD) of 1.4, suggesting that hydrogen transfer is not rate-limiting. 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 electron paramagnetic resonance (EPR). The radical in the reaction of clostridial LAM has the (S)-configuration, whereas that in the reaction of E. coli LAM has the (R)-configuration. Moreover, the conformations of the beta-lysine-related radicals at the active sites of E. coli and clostridial LAM are different. The nuclear hyperfine splitting between the C3 hydrogen and the unpaired electron at C2 shows the dihedral angle to be 6 degrees, unlike the value of 77 degrees reported for the analogous radical bound to the clostridial enzyme. Reaction of (S)-4-thialysine produces a substrate-related radical in the steady state of E. coli LAM, as in the action of the clostridial enzyme. While (S)-beta-lysine is not a substrate for E. coli LAM, it undergoes hydrogen abstraction to form an (S)-beta-lysine-related radical with the same stereochemistry of hydrogen transfer from C2 of (S)-beta-lysine to the 5'-deoxyadenosyl radical as in the action of the clostridial enzyme. The resulting beta-lysyl radical has a conformation different from that at the active site of clostridial LAM. All evidence indicates that the opposite stereochemistry displayed by E. coli LAM is determined by the conformation of the lysine side chain in the active site. Stereochemical models for the actions of LAM from C. subterminale and E. coli are presented.

IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins.

IscR (iron-sulfur cluster regulator) is encoded by an ORF located immediately upstream of genes coding for the Escherichia coli Fe-S cluster assembly proteins, IscS, IscU, and IscA. IscR shares amino acid similarity with MarA, a member of the MarA/SoxS/Rob family of transcription factors. In this study, we found that IscR functions as a repressor of the iscRSUA operon, because strains deleted for iscR have increased expression of this operon. In addition, in vitro transcription reactions established a direct role for IscR in repression of the iscR promoter. Analysis of IscR by electron paramagnetic resonance showed that the anaerobically isolated protein contains a [2Fe-2S](1+) cluster. The Fe-S cluster appears to be important for IscR function, because repression of iscR expression is significantly reduced in strains containing null mutations of the Fe-S cluster assembly genes iscS or hscA. The finding that IscR activity is decreased in strain backgrounds in which Fe-S cluster assembly is impaired suggests that this protein may be part of a novel autoregulatory mechanism that senses the Fe-S cluster assembly status of cells.

A novel lysine 2,3-aminomutase encoded by the yodO gene of bacillus subtilis: characterization and the observation of organic radical intermediates.

The yodO gene product of Bacillus subtilis has been cloned and overexpressed in Escherichia coli and purified. The nucleotide sequence encodes a protein of 471 amino acids with a calculated molecular mass of 54071 Da. The translated amino acid sequence is more than 60% identical to that of the lysine 2,3-aminomutase from Clostridium subterminale SB4. Analytical HPLC gel-permeation chromatography leads to an estimate of an over all molecular mass of 224000+/-21000 Da, which corresponds to a tetrameric protein. The purified protein contains iron, sulphide and pyridoxal 5'-phosphate (PLP) and displays an optical absorption band extending to 700 nm, suggesting the presence of an iron-sulphide cluster. After reductive incubation with L-cysteine anaerobically, the protein catalyses the transformation of L-lysine into beta-lysine in the presence of S-adenosylmethionine (AdoMet) and sodium dithionite. The K(m) value for L-lysine is estimated to be 8.0+/-2.2 mM. The iron-sulphur centre is stable in air,allowing aerobic purification. EPR spectroscopy at 10 K of the purified enzyme revealed an EPR signal similar to that of the [4Fe-4S](3+) cluster observed in the clostridial lysine 2, 3-aminomutase. Incubation with cysteine under anaerobic conditions converts the iron-sulphur centre into the EPR-silent [4Fe-4S](2+). Unlike the clostridial enzyme, the fully reduced [4Fe-4S](+) could not be characterized by further reduction with dithionite in the presence of AdoMet, although both dithionite and AdoMet were required to activate the enzyme. Upon addition of L-lysine, dithionite and AdoMet to the reduced enzyme and freezing the solution to 77 K, the EPR spectrum revealed the presence of an organic free-radical signal (g=2.0023), which displayed multiple hyperfine transitions very similar to the spectrum of the beta-lysine-related radical in the mechanism of the clostridial lysine 2,3-aminomutase. Experiments with isotopically substituted L-lysine and lysine analogues verified the association of spin density with the carbon skeleton of lysine. The data indicate that the protein encoded by the yodO gene of B. subtilis is a novel lysine 2,3-aminomutase. The E. coli homologue of clostridial lysine 2,3-aminomutase was also expressed in E. coli and purified. This protein contained ironand sulphide but not PLP, it did not display lysine 2,3-aminomutase activity, and addition of PLP did not induce 2,3-aminomutase activity.

Lysine 2,3-aminomutase from Clostridium subterminale SB4: mass spectral characterization of cyanogen bromide-treated peptides and cloning, sequencing, and expression of the gene kamA in Escherichia coli.

Lysine 2,3-aminomutase (KAM, EC 5.4.3.2.) catalyzes the interconversion of L-lysine and L-beta-lysine, the first step in lysine degradation in Clostridium subterminale SB4. KAM requires S-adenosylmethionine (SAM), which mediates hydrogen transfer in a mechanism analogous to adenosylcobalamin-dependent reactions. KAM also contains an iron-sulfur cluster and requires pyridoxal 5'-phosphate (PLP) for activity. In the present work, we report the cloning and nucleotide sequencing of the gene kamA for C. subterminale SB4 KAM and conditions for its expression in Escherichia coli. The cyanogen bromide peptides were isolated and characterized by mass spectral analysis and, for selected peptides, amino acid and N-terminal amino acid sequence analysis. PCR was performed with degenerate oligonucleotide primers and C. subterminale SB4 chromosomal DNA to produce a portion of kamA containing 1,029 base pairs of the gene. The complete gene was obtained from a genomic library of C. subterminale SB4 chromosomal DNA by use of DNA probe analysis based on the 1,029-base pair fragment. The full-length gene consisted of 1,251 base pairs specifying a protein of 47,030 Da, in reasonable agreement with 47, 173 Da obtained by electrospray mass spectrometry of the purified enzyme. N- and C-terminal amino acid analysis of KAM and its cyanogen bromide peptides firmly correlated its amino acid sequence with the nucleotide sequence of kamA. A survey of bacterial genome databases identified seven homologs with 31 to 72% sequence identity to KAM, none of which were known enzymes. An E. coli expression system consisting of pET 23a(+) plus kamA yielded unsatisfactory expression and bacterial growth. Codon usage in kamA includes the use of AGA for all 29 arginine residues. AGA is rarely used in E. coli, and arginine clusters at positions 4 and 5, 25 and 27, and 134, 135, and 136 apparently compound the barrier to expression. Coexpression of E. coli argU dramatically enhanced both cell growth and expression of KAM. Purified recombinant KAM is equivalent to that purified from C. subterminale SB4.

Kinetic mechanism of UDP-hexose synthase, a point variant of hexose-1-phosphate uridylyltransferase from Escherichia coli.

Galactose-1-phosphate (galactose-1-P) uridylyltransferase from Escherichia coli catalyzes the interconversion of UDP-glucose and galactose-1-P with UDP-galactose and glucose-1-P by a double-displacement mechanism through a uridylyl-enzyme intermediate, in which the uridine-5'-phosphoryl group is covalently bonded to Nepsilon of His 166. The point variant H166G displays a UDP-hexose synthase activity, in that it catalyzes the reaction of uridine 5'-phosphoimidazolide (UMPIm) with glucose-1-P to form UDP-glucose and imidazole. Inasmuch as the wild-type uridylyltransferase catalyzes its cognate reaction with ping-pong kinetics, an intrinsically ordered substrate binding mechanism, the kinetic mechanism of the UDP-hexose synthase activity of H166G became of interest. The synthase activity follows sequential kinetics [Kim, J., Ruzicka, F., and Frey, P. A. (1990) Biochemistry 29, 10590-10593]. In this work, product inhibition patterns for the synthase activity of H166G indicate random equilibrium binding of substrates. Comparison of the synthase activities of the variants H166G and H166A showed that the glycine variant is about 340- and 600-fold more active than the alanine variant in the forward and reverse directions, respectively. The kinetic consequences of varying the amino acid at position 166 were largely kcat effects, with more modest Km effects. Comparison of the synthase activities of these variants with that of the wild-type enzyme in the production of glucose-1-P showed that the loss of the beta-carbon of His 166 in the complex H166G-UMPIm increases the activation energy for uridylyl group transfer by 2.4 kcal mol-1, and the presence of two additional hydrogen atoms in the complex H166A-UMPIm increases the activation energy by 6.2 kcal mol-1. It is concluded that the active site is much less tolerant of additional steric bulk in the locus of the beta-carbon of His 166 than it is of the loss of the beta-carbon. The sensitivities to additional steric bulk around other positions of the His 166-imidazole ring are much less severe, as indicated by the reactivities of methylated analogues of UMPIm in the synthase reaction of H166G. Uridine 5'-phospho-N-methylimidazolide is more reactive as a synthase substrate than UMPIm, and this is attributed to the positive charge of the imidazole ring. The fact that the imidazole ring of the wild-type covalent uridylyl-enzyme retains its proton and is positively charged is supported by the pH-rate profile for hydrolysis of the intermediate.

S-Adenosylmethionine-dependent reduction of lysine 2,3-aminomutase and observation of the catalytically functional iron-sulfur centers by electron paramagnetic resonance.

Lysine 2,3-aminomutase catalyzes the interconversion of l-alpha-lysine and l-beta-lysine. The enzyme contains an iron-sulfur cluster with unusual properties, and it requires pyridoxal-5'-phosphate (PLP) and S-adenosylmethionine (AdoMet) for activity. The reaction proceeds by a substrate radical rearrangement mechanism, in which the external aldimine formed between PLP and lysine is initially converted into a lysyl-radical intermediate by hydrogen abstraction from C3. The present research concerns the mechanism by which a hydrogen-abstracting species is generated at the active site of lysine 2,3-aminomutase. Earlier tritium tracer experiments have implicated the 5'-deoxyadenosyl moiety of AdoMet in this process. AdoMet is here shown to interact with the iron-sulfur cluster at the active site of Clostridial lysine 2,3-aminomutase. Reduction of the iron-sulfur cluster from its EPR-silent form [4Fe-4S]2+ to the fully reduced form [4Fe-4S]1+ requires the presence of either AdoMet or S-adenosylhomocysteine (SAH) and a strong reducing agent such as dithionite or deazariboflavin and light. The reduced forms are provisionally designated E-[4Fe-4S]1+/AdoMet and E-[4Fe-4S]1+/SAH, and they display similar low-temperature EPR spectra centered at gav = 1.91. The reduced form E-[4Fe-4S]1+/AdoMet is fully active in the absence of any added reducing agent, whereas the form E-[4Fe-4S]1+/SAH is not active. It is postulated that the active form E-[4Fe-4S]1+/AdoMet is in equilibrium with a low concentration of a radical-initiating form that contains the 5'-deoxyadenosyl radical. Initiation of the radical rearrangement mechanism is postulated to take place by action of the 5'-deoxyadenosyl radical in abstracting a hydrogen atom from carbon-3 of lysine, which is bound as its external aldiminine with PLP. This process accounts for the results of tritium tracer experiments, it explains the radical rearrangement mechanism, and it rationalizes the roles of AdoMet and the [4Fe-4S] cluster in the reaction.

Mechanistic roles of tyrosine 149 and serine 124 in UDP-galactose 4-epimerase from Escherichia coli.

Synthesis and overexpression of a gene encoding Escherichia coli UDP-galactose 4-epimerase and engineered to facilitate cassette mutagenesis are described. General acid-base catalysis at the active site of this epimerase has been studied by kinetic and spectroscopic analysis of the wild-type enzyme and its specifically mutated forms Y149F, S124A, S124V, and S124T. The X-ray crystal structure of Y149F as its abortive complex with UDP-glucose is structurally similar to that of the corresponding wild-type complex, except for the absence of the phenolic oxygen of Tyr 149. The major effects of mutations are expressed in the values of kcat and kcat/Km. The least active mutant is Y149F, for which the value of kcat is 0.010% of that of the wild-type epimerase. The activity of S124A is also very low, with a kcat value that is 0.035% of that of the native enzyme. The values of Km for Y149F and S124A are 12 and 21% of that of the wild-type enzyme, respectively. The value of kcat for S124T is about 30% of that of the wild-type enzyme, and the value of Km is similar to that of the native enzyme. The reactivities of the mutants in UMP-dependent reductive inactivation by glucose are similarly affected, with kobs being decreased by 6560-, 370-, and 3.4-fold for Y149F, S124A, and S124T, respectively. The second-order rate constants for reductive inactivation by NaBH3CN, which does not require general base catalysis, are similar to that for the native enzyme in the cases of S124A, S124T, and S124V. However, Y149F reacts with NaBH3CN 12-20-fold faster than the wild-type enzyme at pH 8.5 and 7.0, respectively. The increased rate for Y149F is attributed to the weakened charge-transfer interaction between Phe 149 and NAD+, which is present with Tyr 149 in the wild-type enzyme. The charge-transfer band is present in the serine mutants, and its intensity at 320 nm is pH-dependent. The pH dependencies of A320 showed that the pKa values for Tyr 149 are 6.08 for the wild-type epimerase, 6.71 for S124A, 6.86 for S124V, and 6.28 for S124T. The low pKa value for Tyr 149 is attributed mainly to the positive electrostatic field created by NAD+ and Lys 153 (4.5 kcal mol-1) and partly to hydrogen bonding with Ser 124 (1 kcal mol-1). The pKa of Tyr 149 is the same as the kinetic pKa for the Bronsted base that facilitates hydride transfer to NAD+. We concluded that Tyr 149 provides the driving force for general acid-base catalysis, with Ser 124 playing an important role in mediating proton transfer.

Structural analysis of the H166G site-directed mutant of galactose-1-phosphate uridylyltransferase complexed with either UDP-glucose or UDP-galactose: detailed description of the nucleotide sugar binding site.

Galactose-1-phosphate uridylyltransferase plays a key role in galactose metabolism by catalyzing the transfer of a uridine 5'-phosphoryl group from UDP-glucose to galactose 1-phosphate. The enzyme from Escherichia coli is composed of two identical subunits. The structures of the enzyme/UDP-glucose and UDP-galactose complexes, in which the catalytic nucleophile His 166 has been replaced with a glycine residue, have been determined and refined to 1.8 A resolution by single crystal X-ray diffraction analysis. Crystals employed in the investigation belonged to the space group P2(1) with unit cell dimensions of a = 68 A, b = 58 A, c = 189 A, and beta = 100 degrees and two dimers in the asymmetric unit. The models for these enzyme/substrate complexes have demonstrated that the active site of the uridylyltransferase is formed by amino acid residues contributed from both subunits in the dimer. Those amino acid residues critically involved in sugar binding include Asn 153 and Gly 159 from the first subunit and Lys 311, Phe 312, Val 314, Tyr 316, Glu 317, and Gln 323 from the second subunit. The uridylyltransferase is able to accommodate both UDP-galactose and UDP-glucose substrates by simple movements of the side chains of Glu 317 and Gln 323 and by a change in the backbone dihedral angles of Val 314. The removal of the imidazole group at position 166 results in little structural perturbation of the polypeptide chain backbone when compared to the previously determined structure for the wild-type enzyme. Instead, the cavity created by the mutation is partially compensated for by the presence of a potassium ion and its accompanying coordination sphere. As such, the mutant protein structures presented here represent valid models for understanding substrate recognition and binding in the native galactose-1-phosphate uridylyltransferase.

Kinetic characterization of an organic radical in the ascarylose biosynthetic pathway.

The lipopolysaccharide of Yersinia pseudotuberculosis V includes a 3,6-dideoxyhexose, ascarylose, as the nonreducing end of the O-antigen tetrasaccharide. The C-3 deoxygenation of CDP-6-deoxy-L-threo-D-glycero-4-hexulose is a critical reaction in the biosynthesis of ascarylose. The first half of the reaction is a dehydration catalyzed by CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which is PMP-dependent and contains a redox-active [2Fe-2S] center. The second half is a reduction that requires an additional enzyme, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3, formerly known as CDP-6-deoxy-delta 3,4-glucoseen reductase), which has a FAD and a [2Fe-2S] center in the active site. Using NADH as the reductant in the coupled E1-E3 reaction, we have monitored the kinetics of a radical intermediate using both stopped-flow spectrophotometry and rapid freeze-quench EPR under aerobic and hypoxic conditions. In the EPR studies, a sharp signal at g = 2.003 was found to appear at a rate which is kinetically competent, reaching its maximum intensity at approximately 150 ms. Stopped-flow UV-vis analysis of the reaction elucidated a minimum of six optically distinguishable states in the mechanism of electron transfer from NADH to substrate. Interestingly, one of the detected intermediates has a time course nearly identical to that of the radical detected by rapid freeze-quench EPR. The difference UV-vis spectrum of this intermediate displays a maximum at 456 nm with a shoulder at 425 nm. Overall, these results are consistent with an electron transfer pathway that includes a radical intermediate with the unpaired spin localized on the substrate-cofactor complex. Evidence in support of this mechanism is presented in this report. These studies add the PMP-glucoseen radical to the growing list of mechanistically important bioorganic radical intermediates that have recently been discovered.

Standard free energies for uridylyl group transfer by hexose-1-P uridylyltransferase and UDP-hexose synthase and for the hydrolysis of uridine 5'-phosphoimidazolate.

The reversible reaction of UDP-glucose with imidazole (Im) to produce uridine 5'-phoshoimidazolate (UMPIm) and glucose-1-P is catalyzed by UDP-hexose synthase, which is the mutant H166G of hexose-1-P uridylyltransferase (EC 2.7.7.12) [Kim, J., Ruzicka, F.J., & Frey, P.A. (1990) Biochemistry 29, 10590-10593]. The availability of UDP-hexose synthase allows the equilibrium constant for the reaction UDP-glucose + Im = UMPIm + glucose-1-P to be measured, and it is found to be 2.2 x 10(-2) at pH 8.5 and 27 degrees C. At pH 7.0 and 27 degrees C the equilibrium constant is 6.4 x 10(-4). The equilibrium constant for the formation of the covalent uridylyl-enzyme intermediate of hexose-1-P uridylyltransferase (E-His(166) + UDP-glucose = E-His(166)-UMP + glucose-1-P) is found to be 1.8 x 10(-4) at pH 7.0 and 25 degrees C, which is slightly less favorable than the formation of UMPIm from UDP-glucose and Im. These equilibrium constants, when considered in the light of other data in the literature, allow the standard free energy changes for the hydrolysis of UMPIm and the analogous covalent uridylyl-enzyme intermediate to be calculated. The results show that delta G' degrees (delta G degrees (ph)(7.0)) for the hydrolyses of UMPIm and E-His(166)-UMP are -14.7 and -15.4 kcal mol(-1), respectively at pH 7.0. At pH 8.5, the corresponding values of delta G degrees (ph) (8.5) are -12.6 and -9.9 kcal mol(-1), respectively. It is concluded that noncovalent binding interactions between the active site and the UMP group of E-His(166)-UMP provide little or no stabilization in the formation of this species as an intermediate in the reaction of hexose-1-P uridylyltransferase.

Galactose-1-phosphate uridylyltransferase from Escherichia coli, a zinc and iron metalloenzyme.

Galactose-1-P uridylyltransferase purified from Escherichia coli cells grown in enriched medium contains approximately 1.2 mol of tightly bound zinc/mol of subunits as well as variable amounts of iron, up to 0.7 mol/mol of subunits, and no detectable Ca, Cd, Cu, Mo, Ni, Co, Mn, As, Pb, or Se. The chelators, 1,10-phenanthroline, 8-hydroxyquinoline, 8-hydroxyquinoline sulfonate, and 2,2'-bipyridyl remove metal ions from the enzyme and allow the importance of zinc and iron to be evaluated. Dialysis of this enzyme against 2 mM 1,10-phenanthroline, 8-hydroxyquinoline sulfonate, and 2,2'-bipyridyl at millimolar concentrations slowly removes both zinc and iron from the enzyme (t1/2 = 4 days at 24 degrees C) with concomitant loss of enzymatic activity. In chelation experiments utilizing 1,10-phenanthroline, residual enzymatic activity was found to be proportional to the zinc content, to the iron content, and to the sum of zinc and iron. UDP-glucose (0.35 mM) protects the enzyme against loss of metal ions and activity in the presence of 1,10-phenanthroline, whereas glucose-1-P at 70 mM (400 x Km) fails to protect. The enzyme purified from cells grown on a minimal medium containing inorganic salts and glucose supplemented with either ZnSO4 or FeSO4 shows approximately the same level of enzymatic activity as the enzyme from cells grown on enriched medium. These experiments showed that enzymatic activity is supported by either iron or zinc associated with two sites in the enzyme. Enzyme depleted of metal ions by chelators can be partially reactivated by addition of ZnSO4.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of iron-sulfur clusters in lysine 2,3-aminomutase by electron paramagnetic resonance spectroscopy.

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)

Metal cofactors of lysine-2,3-aminomutase.

Lysine-2,3-aminomutase from Clostridium SB4 contains iron and sulfide in equimolar amounts, as well as cobalt, zinc, and copper. The iron and sulfide apparently constitute an Fe-S cluster that is required as a cofactor of the enzyme. Although no B12 derivative can be detected, enzyme-bound cobalt is a cofactor; however, the zinc and copper bound to the enzyme do not appear to play a role in its catalytic activity. These conclusions are supported by the following facts reported in this paper. Purification of the enzyme under anaerobic conditions increases the iron and sulfide content. Lysine-2,3-aminomutase purified from cells grown in media supplemented with added CoCl2 contains higher levels of cobalt and correspondingly lower levels of zinc and copper relative to enzyme from cells grown in media not supplemented with cobalt. The specific activity of the purified enzyme increases with increasing iron and sulfide content, and it also increases with increasing cobalt and with decreasing zinc and copper content. The zinc and copper appear to occupy cobalt sites under conditions of insufficient cobalt in the growth medium, and they do not support the activity of the enzyme. The best preparations of lysine-2,3-aminomutase obtained to date exhibit a specific activity of approximately 23 units/mg of protein and contain about 12 g atoms of iron and of sulfide per mol of hexameric enzyme. These preparations also contain 3.5 g atoms of cobalt per mol, but even the best preparations contain small amounts of zinc and copper. The sum of cobalt, zinc, and copper in all preparations analyzed to date corresponds to 5.22 +/- 0.75 g atoms per mol of enzyme. An EPR spectrum of the enzyme as isolated reveals a signal corresponding to high spin Co(II) at temperatures below 20 K. The signal appears as a partially resolved 59Co octet centered at an apparent g value of 7. The 59Co hyperfine splitting (approximately 35 G) is prominent at 4.2 K. These findings show that lysine-2,3-aminomutase requires Fe-S clusters and cobalt as cofactors, in addition to the known requirement for pyridoxal 5'-phosphate and S-adenosylmethionine.

Modulation of interferon receptor expression during combination beta ser-interferon and gamma-interferon treatment of human colon carcinoma cells.

Combination treatment of SKCO1 human colon carcinoma cells with beta ser-interferon (IFN-beta ser) and gamma-interferon (IFN-gamma) results in a synergistic antiproliferative effect. The role of IFN-beta ser and IFN-gamma receptor modulation was investigated as a possible mechanism for this response. IFN-gamma (0.05-50 ng/ml) pretreatment of SKCO1 cells for 24 h decreased specific binding of 125I-IFN-beta ser by 35-60%. Scatchard analysis of binding data obtained following 24-h treatment with 5 ng/ml IFN-gamma showed that this reduction in binding was due to a decreased receptor affinity (control cells, Kd = 46 +/- 1.6 pM; IFN-gamma-treated cells, Kd = 106 +/- 6 pM, n = 2) rather than a significant change in receptor number (receptor number/control cell = 1214 +/- 471, receptor number/IFN-gamma treated cell = 1118 +/- 153, n = 2). In contrast, pretreatment of SKCO1 cells with IFN-beta ser (5 ng/ml) resulted in slight (10-35%) increases in 125I-IFN-gamma-specific binding. Scatchard analysis of binding data obtained following 24-h treatment with 5 ng/ml IFN-beta ser showed a decrease in binding affinity (control cells, Kd = 28 +/- 7 pM; IFN-beta ser-treated cells, Kd = 38 +/- 7 pM, n = 2) and a 32% increase in IFN-gamma receptor sites (receptor number/control cell = 4257 +/- 464, receptor number/IFN-beta ser-treated cell = 5570 +/- 730; n = 2). 125I-IFN-gamma internalization studies performed at 37 degrees C confirmed the cell surface binding assays; IFN-beta ser-treated cells internalized 30-50% more labeled IFN-gamma than untreated cells. However, it is unlikely that differences in binding and internalization of this magnitude play a primary role in the synergistic antiproliferative effect of IFN-gamma with IFN-beta ser in SKCO1 cells. Biochemical modulation at sites distal to the ligand receptor interaction should be investigated.

Binding of recombinant-produced interferon beta ser to human lymphoblastoid cells. Evidence for two binding domains.

Human interferon beta (IFN beta ser), produced by recombinant DNA technology, was radiolabeled to approximately one atom of iodine-125/molecule of interferon without detectable loss of antiviral activity. At 37 degrees C, binding of 125I IFN beta ser occurred rapidly (t1/2max less than or equal to 15 min) followed by internalization and degradation of bound ligand. Kinetic analysis at 4 degrees C indicated diffusion-limited association kinetics independent of 125I IFN beta ser concentration. Dissociation of bound 125I IFN beta ser from Daudi cells was slow (t1/2 = 1.2 h) of bound radiolabeled ligand was observed in the presence of unlabeled IFN beta ser, naturally produced IFN beta, and IFN alpha 6, but was not observed with unlabeled IFN gamma or nonspecific proteins. Concomitantly, equilibrium analysis indicated heterogeneous binding of 125I IFN beta ser to six cell lines of lymphoid origin consistent with either negative cooperativity or two populations of receptors. Analysis of binding of 125I IFN beta ser to Daudi cell receptors in the presence of unlabeled IFN alpha 6 suggested that one receptor served both ligands. The latter conclusion was supported by results of chemical cross-linking experiments in which an 125I IFN beta ser/receptor complex (Mr 120,000-130,000) was observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This complex was absent when binding occurred in the presence of either excess unlabeled IFN beta ser or IFN alpha 6.

Variation in the binding of 125I-labeled interferon-beta ser to cellular receptors during growth of human renal and bladder carcinoma cells in vitro.

Studies of various established human bladder and renal carcinoma cell lines cultured in vitro demonstrated the presence of specific, saturable, high affinity binding sites for 125I-labeled human interferon Beta ser IFN-beta ser). This recombinant produced interferon labeled with approximately one atom of 125I/molecule of IFN expressed minimal or no loss of antiviral activity. A single class of binding sites (1000-2000/cell) with an affinity constant of 10(10)-10(11) L/M was measured at 4 degrees C for cells exhibiting widely different sensitivity to the antiproliferative effect of IFN-beta ser. Major fluctuations in the binding of 125I-labeled IFN-beta ser to cellular receptors were observed during in vitro proliferation of four of five cell lines examined. A significant decrease (P less than 0.001) in specific binding was observed 48 h after cultures were established. Cell cycle analysis suggested that within the first 24 h and in the very late log and stationary phase of growth of ACHN (human renal carcinoma) cells, variations in the binding of 125I-labeled IFN-beta ser were partially attributable to binding fluctuations during the mitotic cycle. The 2- to 3-fold decline 24 h following plating of ACHN cells corresponded to a 70% decrease in the number of cells in G0-G1. T24 (human transitional cell carcinoma) and ACHN cells, synchronized by serum starvation, demonstrated increased binding of 125I-labeled IFN-beta ser 4-16 h following serum replenishment. This increase in receptor binding occurred prior to the onset of DNA and protein synthesis and was followed by a decline immediately prior to cell division. Binding site analysis indicated that the increased binding prior to DNA synthesis was due to a 5- to 6-fold increase in receptor affinity for the radiolabeled ligand. After an initial 40% decline in receptors per cell following serum stimulation, receptor concentration remained essentially unchanged. Induction of 2',5'-oligoadenylate synthetase in ACHN cells and antiproliferative activity in RT112, RT4, T24 (human transitional cell carcinoma), and ACHN cells by IFN-beta ser decreased significantly 48 h following plating. These changes in the biological activity of this interferon corresponded to growth related fluctuations in the IFN-beta ser binding.

Nuclear thyroid hormone receptors, alpha-glycerophosphate dehydrogenases, and malic enzyme in N-nitrosomethylurea-induced rat mammary tumors.

Specific L-3,3'5-triiodothyronine (triiodothyronine) binding by isolated nuclei was determined in N-nitrosomethylurea-induced mammary carcinomas and livers from intact and thyroidectomized animals. Tumors contained a single class of high-affinity nuclear triiodothyronine binding sites with characteristics similar to those of hepatic nuclei. Competition experiments showed the relative affinities of thyroid hormone structural analogues for both tumor and liver receptors to be: triiodothyroacetic acid greater than triiodothyronine greater than thyroxine greater than reverse triiodothyronine. Diiodotyrosine did not complete for the binding sites. Mammary tumors exhibited a wide range of binding sites, but most were in the range of 50 to 150 fmol/micrograms DNA. The levels were not affected significantly by hypothyroidism. Liver triiodothyronine receptor concentrations were approximately 5-fold those of tumors; they were unaffected by low serum thyroid hormone levels and were similar to those of non-tumor-bearing, euthyroid controls. Mitochondrial alpha-glycerophosphate dehydrogenase and cytosolic malic enzyme activities were reduced in both tumors and livers of hypothyroid rats; cytosolic alpha-glycerophosphate dehydrogenase levels were unchanged. Hepatic enzyme activities were similar in euthyroid tumor-bearing animals and in euthyroid healthy controls.

The influence of thyroidal status on rat hepatic mitochondrial NADH duroquinone reductase.

Hepatic mitochondrial NADH duroquinone reductase and alpha-glycerophosphate dehydrogenase activities were measured in rats with altered thyroidal status. Whereas alpha-glycerophosphate dehydrogenase activity was decreased in hypothyroid rats, NADH duroquinone reductase was increased approximately 3-fold in both thiouracil-fed and thyroidectomized rats. In hyperthyroid animals, NADH duroquinone reductase activity was decreased, whereas there was the expected elevation in mitochondrial alpha-glycerophosphate dehydrogenase. Maximum velocity measurements of NADH duroquinone reductase demonstrated that the increase in enzyme activity associated with hypothyroidism occurred without an alteration in Michaelis constants for the reaction. Rats bearing mammary carcinomas induced by N-nitrosomethylurea also showed an increase in hepatic NADH duroquinone reductase when rendered hypothyroid, but the enzyme was unaffected by thyroxine administration.

The high potential iron-sulfur cluster of aconitase is a binuclear iron-sulfur cluster.

It has been reported (Ruzicka, F.J., and Beinert, H. (1978) J. Biol. Chem. 253, 2514-2517) that aconitase in the oxidized state, as isolated, shows an electron paramagnetic resonance signal centered at g = 2.01, typical of high potential iron-sulfur proteins. Since the magnetic state corresponding to this signal has thus far only been found in tetranuclear iron-sulfur clusters in model compounds and proteins, it could be expected that aconitase also contains a [4Fe-4S] cluster. We show here that core extrusion, in the presence of hexamethylphosphoramide and o-xylyl-alpha,alpha'-dithiol and subsequent ligand exchange with p-trifluoromethylbenzenethiol yield absorption spectra typical of binuclear iron-sulfur clusters. According to the absorbance measured, the concentration of the extruded [2Fe-2S] cluster quantitatively accounts for the iron-sulfur content of the preparations examined. Preliminary studies of the 19F nuclear magnetic resonance spectrum obtained on extrusion with p-trifluoromethylbenzenethiol confirm the presence of a binuclear cluster in aconitase.