Physico-chemical characteristics of the new antitumor antibiotic virenomycin

Virenomycin, a new crystalline antitumor antibiotic was isolated from the mycelium of Streptomyces virens. The antibiotic contained: C 64.87 per cent, H 5.66 per cent, methoxylic groups 9.5 per cent. The melting temperature was 255-260 degrees (dec.), [alpha]20D=-17 (c 0.142, chloroform). Virenomycin had a complex UV spectrum with lambdamax. 245 (677), 265 (453), 275 (542), 287 (507), 395 (222) nm. A chromofor fragment and carbohydrate (C7H14O5) were found in the methanolysis products. Virenomycin was close to antibiotic c B-21085 BY THe physico-chemical properties and differed from it in the character of the UV spectrum and the values of the specific absorption, as well as by the optic rotation in dimethyl sulphoxide and acetic acid.     

Isolation of the new antibiotic variamycin B from the biosynthetic products of a Streptomyces olivovariabilis sp. nov. culture

Variamycin B, a new antitumor antibiotic was isolated from metabolic products of Streptomyces olivovariabilis sp. nov. The empirical formula of variamycin B is C52H76O24, the melting point is 163-165 degrees C (dec.), (alpha)20D--30 +/- 2 degrees (C 0.5, alcohol), the UV-spectrum: lambda max 230, 279, 317, 330, 415 (lg E 4.27, 4.6, 3.83, 3.75, 3.95), IR-spectrum: 1080, 1170, 1380, 1570, 1640, 1720, 3400 cm-1. Variamycin B is classified as belonging to the group of antibiotic analogs of aureolic acid. Chromomycinone, tetrazide and residues of two 2,6-didesoxysugars, i.e. olivose and variose were obtained as a result of complete and partial acid degradation of variamycin B. It was shown with the data of quantitative analysis of the hydrolysates obtained on complete hydrolysis of variamycin B that the ratio between the residues of variose and olivose in the antibiotic molecule was 1 : 4. Oxidation of variamycin B with Fremi salt resulted in formation of chinone having chromomycinone-chinone, 2 residues of olivose and 1 residue of variose in its composition. Investigation of the products of partial hydrolysis provided isolation of a substance having aglicone-chromomycenone and 4 residues of olivose in its composition. A partial structure of variamycin B is proposed.     

Purification of the yellow fluorescent protein from Vibrio fischeri and identity of the flavin chromophore

A low molecular weight protein (approximately 25,000 D) exhibiting a yellow fluorescence emission peaking at approximately 540 nm was isolated from Vibrio fischeri (strain Y-1) and purified to apparent homogeneity. FMN is the chromophore, but it exhibits marked red shifts in both the absorption (lambda max = 380, 460 nm) and the fluorescence emission. When added to purified luciferase from the same strain, which itself catalyzes an emission of blue-green light (lambda max approximately 495 nm), this protein induces a bright yellow luminescence (lambda max approximately 540 nm); this corresponds to the emission of the Y-1 strain in vivo. This yellow bioluminescence emission is thus ascribed to the interaction of these two proteins, and to the excitation of the singlet FMN bound to this fluorescent protein.     

Role of histidine 42 in ascorbate peroxidase. Kinetic analysis of the H42A and H42E variants.

To examine the role of the distal His42 residue in the catalytic mechanism of pea cytosolic ascorbate peroxidase, two site-directed variants were prepared in which His42 was replaced with alanine (H42A) or glutamic acid (H42E). Electronic spectra of the ferric derivatives of H42A and H42E (pH 7.0, mu = 0.10 m, 25.0 degrees C) revealed wavelength maxima [lambda(max) (nm): 397, 509, approximately equal to 540(sh), 644 (H42A); 404, 516, approximately equal to 538(sh), 639 (H42E)] consistent with a predominantly five-co-ordinate high-spin iron. The specific activity of H42E for oxidation of L-ascorbate (8.2 +/- 0.3 U.mg(-1)) was approximately equal to 30-fold lower than that of the recombinant wild-type enzyme (rAPX); the H42A variant was essentially inactive but activity could be partially recovered by addition of exogenous imidazoles. The spectra of the Compound I intermediates of H42A [lambda(max) (nm) = 403, 534, 575(sh), 645] and H42E [lambda(max) (nm) = 404, 530, 573(sh), 654] were similar to those of rAPX. Pre-steady-state data for formation of Compound I for H42A and H42E were consistent with a mechanism involving accumulation of a transient enzyme intermediate (K(d)) followed by conversion of this intermediate into Compound I (k'(1)). Values for k'(1) and K(d) were, respectively, 4.3 +/- 0.2 s(-1) and 30 +/- 2.0 mM (H42A) and 28 +/- 1.0 s(-1) and 0.09 +/- 0.01 mM (H42E). Photodiode array experiments for H42A revealed wavelength maxima for this intermediate at 401 nm, 522 nm and 643 nm, consistent with the formation of a transient [H42A-H(2)O(2)] species. Rate constants for Compound I formation for H42A were independent of pH, but for rAPX and H42E were pH-dependent [pKa = 4.9 +/- 0.1 (rAPX) and pK(a) = 6.7 +/- 0.2 (H42E)]. The results provide: (a) evidence that His42 is critical for Compound I formation in APX; (b) confirmation that titration of His42 controls Compound I formation and an assignment of the pK(a) for this group; (c) mechanistic and spectroscopic evidence for an intermediate before Compound I formation; (d) evidence that a glutamic acid residue at position 42 can act as the acid-base catalyst in ascorbate peroxidase.     

Mycosporine-like amino acid (MAAs) production by Heterocapasa sp. (Dinophyceae) in indoor cultures

The possibility of using mycosporine-like amino acids (MAAs), with an apparent sunscreen function in nature, as ultraviolet radiation (UVR) blockers to prevent skin injury has been raised by diverse authors. Production of MAAs by the dinoflagellate Heterocapsa sp. (Dinophyceae) is shown here. Three major peaks with absorption maxima at 330.8, 332.0 and 333.2 nm were detected by high performance liquid chromatography (HPLC) analysis of methanolic extracts in all tested conditions. Analysis of crude extract by mass spectroscopy with electrospray ionization (MS-EI) showed a set of molecular ions ([M+H](+)) with main peaks being at m/z 242.4, 288.4, 303.3 and 333.3 u.m.a. According to these data, along with retention times, the MAA profile of Heterocapsa sp. is assumed to be composed of shinorine (lambda(max)=334 nm), mycosporine-2-glycine (lambda(max)=331 nm) and palythinol (lambda(max)=332 nm). A constitutive MAA content of about 4 microg (10(6) cells)(-1) was measured under exposure to PAR only. A maximal accumulation of MAA per culture volume of 1.1 mg l(-1) was obtained after 72 h of exposure to PAR+UVA, while the highest production rate (0.025 mg l(-1) h(-1)) was computed after 24 h of exposure to PAR+UVA+UVB.     

Fermentation, isolation, and biological activity of maduramycin: a new antibiotic from Actinomadura rubra.

In a continuing search for new antibiotics, the species Actinomadura rubra (Sveshnikova et al.) J. Meyer et M. Sveshnikova 1974 (strain IMET 13001) was found to produce a red pigment with indicator properties, designated maduramycin. The pigment (C28H22O10; m.w. 518 m/e-; m.p. 305--310 degrees C (dec.); UVmax 225.307 nm) possesses a strong antimicrobial activity against gram-positive bacteria, including strains which produce inactivating enzymes for some commercial antibiotics. Maduramycin forms a complex with serum albumin, but no complex formation with DNA was observed using absorption spectroscopic and polarographic methods. Maduramycin additionally inhibits the action of some enzymes. The LC50 of maduramycin in mice was greater than 250 mg/kg on intraperitoneal administration. Fermentation, isolation, and some of the chemical and biological properties of this new antibiotic are described.     

Detection and identification of intermediates in the reaction of L-serine with Escherichia coli tryptophan synthase via rapid-scanning ultraviolet-visible spectroscopy

Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the UV-visible absorption changes (300-550 nm) that occur in the spectrum of enzyme-bound pyridoxal 5'-phosphate during the reaction of L-serine with the alpha 2 beta 2 and beta 2 forms of Escherichia coli tryptophan synthase. In agreement with previous kinetic studies [Lane, A., & Kirschner, K. (1983) Eur. J. Biochem. 129, 561-570], the reaction with alpha 2 beta 2 was found to occur in three detectable relaxations (1/tau 1 greater than 1/tau 2 greater than 1/tau 3). The RSSF data reveal that during tau 1, the internal aldimine, E(PLP), with lambda max = 412 nm (pH 7.8), undergoes rapid conversion to two transient species, one with lambda max congruent to 420 nm and one with lambda max congruent to 460 nm. These species decay in a biphasic process (1/tau 2, 1/tau 3) to a complicated final spectrum with lambda max congruent to 350 nm and with a broad envelope of absorbance extending out to approximately 525 nm. Analysis of the time-resolved spectra establishes that the spectral changes in tau 2 are nearly identical with the spectral changes in tau 3. Kinetic isotope effects due to substitution of 2H for the alpha-1H of serine were found to increase the amount of the 420-nm transient and to decrease the amount of the species with lambda max congruent to 460 nm. These findings identify the serine Schiff base (the external aldimine) as the 420 nm absorbing, highly fluorescent transient; the species with lambda max congruent to 460 nm is the delocalized carbanion (quinoidal) species derived from abstraction of the alpha proton from the external aldimine. The reaction of L-serine with beta 2 consists of two relaxations (1/tau 1 beta greater than 1/tau 2 beta) and yields a quasi-stable species with lambda max = 420 nm, in good agreement with a previous report [Miles, E. W., Hatanaka, M., & Crawford, I. P. (1968) Biochemistry 7, 2742-2753]. Analysis of the RSSF spectra indicates that the same spectral change occurs in each phase of the reaction. The similarity of the spectral changes that occur in tau 2 and tau 3 of the alpha 2 beta 2 reaction is postulated to originate from the existence of two (slowly) interconverting forms of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Color vision of the coelacanth (Latimeria chalumnae) and adaptive evolution of rhodopsin (RH1) and rhodopsin-like (RH2) pigments

The coelacanth, a "living fossil," lives at a depth of about 200 m near the coast of the Comoros archipelago in the Indian Ocean and receives only a narrow range of light at about 480 nm. To see the entire range of "color" the Comoran coelacanth appears to use only rod-specific RH1 and cone-specific RH2 visual pigments, with the optimum light sensitivities (lambda max) at 478 nm and 485 nm, respectively. These blue-shifted lambda max values of RH1 and RH2 pigments are fully explained by independent double amino acid replacements E122Q/A292S and E122Q/M207L, respectively. More generally, currently available mutagenesis experiments identify only 10 amino acid changes that shift the lambda max values of visual pigments more than 5 nm. Among these, D83N, E1220, M207L, and A292S are associated strongly with the adaptive blue shifts in the lambda max values of RH1 and RH2 pigments in vertebrates.     

Physicochmemical characteristics of the new antineoplastic antibiotic, nocamycin]

Nocamycin is produced by Nocardiopsis syringae. It is recovered from the culture fluid by extraction with chloroform. The molecular weight of the crystalline antibiotic is 503, its melting point is 147--149 degrees, [alpha]20 degrees D = --50 degrees (c. 0.21, chloroform), lambdamax235 and 348 nm(E1%sm= = 150 and 420), the summation formula is C26 H33NO9, the biological activity is 100000 Units/mg with respect to Bacillus mycoides. Nocamycin forms salts with alkalies soluble in water. On hydrolysis with an alkali it forms carbonic acid having no ester bond (IP spectrum) and methoxylic group present in the antibiotic molecule. Nocamycin is a new natural substance.     

Antibacterial colorants: characterization of prodiginines and their applications on textile materials

A strain of Vibrio sp. isolated from marine sediments produced large quantities of bright red pigments that could be used to dye many fibers including wool, nylon, acrylics, and silk. Characterization of the pigments by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) revealed three prodiginine-like structures with nonpolar characteristics and low molecular mass. UV-visible spectra of the major constituent in methanol solution showed absorbance at lambda max 530 nm wavelength. The accurate mass result showed that the main isolated product has a molecular mass of m/z 323.1997. Further analysis using mass fragmentation (MS/MS), 1H NMR, COSY, HMQC NMR and DEPT confirmed the detailed structure of the pigment with an elementary composition of C20H25N3O. Fabrics dyed with the microbial prodiginines demonstrated antibacterial activity.     

Polarized absorption spectra of green fluorescent protein single crystals: transition dipole moment directions

Polarized absorption spectra of orthorhombic crystals of wild-type green fluorescent protein (GFP) were measured between 350 and 520 nm to obtain information on the directions of the electronic transition dipole moments ((-->)m) of the chromophore relative to the molecular axes. The transition dipole moment orientation is a basic spectroscopic parameter of relevance to biologists when interpreting F?rster-type fluorescence resonance energy transfer data and for comparing absorbance and fluorescence spectra of GFP with quantum chemical calculations. Maximal extinction was obtained throughout the spectrum when the polarization direction of the electric vector of incident light was parallel to the c-axis of the crystal. The transition dipole moments were assumed to be parallel to the plane of the chromophore. With this assumption and the measured dichroic ratios in the crystals, the transition dipole moments associated with the neutral (lambda(max) = 398 nm) and anionic (lambda(max) = 478 nm) forms of the chromophore were found to subtend angles of approximately 26 degrees and 13 degrees (counterclockwise), respectively, with the vector that joins the phenolic and imidazolinone oxygen atoms of the chromophore.     

Isoepoxydon, a new metabolite of the patulin pathway in Penicillium urticae

A patulin-negative mutant (J1) of Penicillium urticae (N.R.R.L. 2159A) was known to accumulate about 100mg per litre quantities of the 5,6-epoxygentisyl quinone, (-)-phyllostine and another metabolite (UIII). Both were derived from acetate and hence were polyketides. Purified UIII (m.p. 53 degrees C, [alpha](32) (D)+206 degrees , lambda(methanol) (max.) 240nm; epsilon 3806 litre.mol(-1).cm(-1)) was characterized as a partially reduced derivative of (-)-phyllostine and was found to be a diastereoisomer of the known phytotoxin, (+)-epoxydon. Hence its designation as (+)-iso- or epi-epoxydon. From (1)H n.m.r. and c.d. data the stereochemistry of the epoxide ring in (+)-isoepoxydon was determined to be identical with that in (+)-epoxydon (i.e. R,R) but the configuration of the secondary alcohol at C-4 was S rather than R as in (+)-epoxydon. Isoepoxydon (compound UIII) is therefore (4S,5R,6R)-5,6-epoxy-4-hydroxy-2-hydroxymethylcyclohex-2-en-1-one. The boat conformation in which the C-4 hydroxy group is axial is preferred. In the range of 1mm to 5mm, the antibiotic activity of (+)-isoepoxydon against Bacillus subtilis sp. was 56% of that obtained with patulin. Over a period of 1 to 3h, [(14)C]isoepoxydon was efficiently converted into patulin by a shake culture of the parent strain of P. urticae. The precursor relationship of isoepoxydon to patulin was confirmed by feeding unlabelled isoepoxydon (1mm) to a washed-cell suspension of a mutant (J2) in which, over a period of 3 to 5h, a better than 60% conversion into patulin was attained. The enzymic relationship between isoepoxydon and phyllostine and their positions in the late portion of the patulin biosynthetic pathway are discussed.     

A correlation between the cellular composition of the spleen and the change in splenocyte chemiluminescence after laser irradiation]

Chemiluminescence (CL) of splenocytes of A/Sn mice (1.5-9-month old) was recorded after irradiation of the cells with lambda semiconductor laser at 820 nm (dose 1.1 x 10(3) J/m2, pulse repetition rate 292 Hz). Laser radiation was found to stimulate or suppress the spontaneous CL (SCL) of splenocytes, the amplitude and its sign depending on cellular composition of the spleen. Direct correlations between effect of laser radiation (per cent in changes in SCL) and per cent of plasmatic cells (r = 0.743, p < 0.001), neutrophils (r = 0.650, p < 0.001) and myelocytes and metamyelocytes (r = 0.507, p < 0.01) were established. The correlation with per cent of lymphocytes (r = -0.590, p < 0.001) was found to be a reverse one.     

Bovine alpha-fetoprotein. Isolation and characterization.

Bovine AFP was purified by ion exchange chromatograph on C.M. cellulose and DEAE Sephadex A-50, gel filtration and immunosorbent technique. AFP was homogeneous when studied by gel electrophoresis under non denaturing and denaturing conditions, by ultracentrifugation and by immunological methods. The following molecular data were obtained: 1. Sedimentation equilibrium indicated a molecular weight of 66,500 and sedimentation velocity gave s degrees 20, w = 4.71 S. A partial specific volume v = 0.737 ml g-1 was derived from density measurements. 2. From these data, a Stokes radius of 3.26 nm, a diffusion coefficient D20 w = 6.61 10(-7) cm2 sec-1 and a frictional ratio f/fo = 1.21 were calculated. 2. Sodium dodecylsulphate disc electrophoresis suggests a molecular weight of 67,000. 3. Gel filtration pointed to a molecular weight of 75,000. 4. Microheterogeneity of AFP was demonstrated by isoelectric focusing. The isoelectric point of the major component is 4.6. 5. The chemical composition was determined. AFP is a glycoprotein containing 7 per cent carbohydrate including 1.67 per cent hexoses, 2.38 per cent N-acetyl glucosamine and 1.8 per cent N-acetyl neuraminic acid.     

Transformation and mineralization of 2,4,6-trinitrotoluene by the white rot fungus<Emphasis Type="Italic"> Irpex lacteus</Emphasis>

Unlike other 2,4,6-trinitrotoluene (TNT)-degrading white rot fungi, including Phanerochaete chrysoporium, initial metabolism of TNT by Irpex lacteus was found to occur through two different transformation pathways. Metabolites of the nitro group reduction pathway were confirmed with the standard compounds, and the formation of hydride-Meisenheimer complex of TNT (H(-)-TNT) formed in the denitration pathway was identified with LC/MS and by LC/photodiode array (PDA) detection. The molecular weight of the H(-)-TNT complex was identified as 228 m/z, and the UV-visible absorption spectrum, recorded with a PDA detector, proved the identity of this metabolite (RT, 18.7 min; lambda(max) 254, 474, 557 nm) by comparison with the authentic synthetic H(-)-TNT (RT 18.7 min; lambda(max) 261, 474, 563 nm). Mineralization of [U-(14)C]TNT by I. lacteus was also measured in static and shaken cultures. The mineralization rate of TNT in the static culture was higher than that in the shaken culture, and addition of Tween 80 (final concentration 1%) enhanced the mineralization of TNT in the static culture, resulting in 30.57% of CO(2) evolution from the radiolabeled TNT added. The high TNT mineralization capability of I. lacteus seemed to be the result of simultaneous utilization of the denitration pathway, which is more favorable for the ring cleavage and mineralization of TNT, together with the nitro group reduction pathway.     

Studies on the interaction of Escherichia coli agmatinase with manganese ions: structural and kinetic studies of the H126N and H151N variants

The H126N and H151N variants of Escherichia coli agmatinase (EC 3.5.3.11) were produced by site-directed mutagenesis, and their kinetic and structural properties were examined. About 51% and 30% of wild-type activity were expressed by fully manganese activated species of the H126N and H151N variants, respectively. Mutations were not accompanied by changes in the K(m) value for arginine (1.2+/-0.3 mM), K(i) value for putrescine inhibition (3.2+/-0.4 mM), molecular weight (M(r) 67,000+/-2000), tryptophan fluorescence properties (lambda(max) = 342 nm) or CD spectra of the enzyme. However, the interaction with the required manganese ions was significantly altered, as indicated by the effects of dialysis of the enzymes against metal-free buffer. We conclude that replacement of His151 with asparagine results in the loss of a catalytically essential Mn(2+) upon dialysis and concomitant reversible inactivation of the H151N mutant, and that the affinity of a more weakly bound Mn(2+) is decreased in the H126N variant.     

Transfer of class 1 integron-mediated antibiotic resistance genes from shiga toxin-producing Escherichia coli to a susceptible E. coli K-12 strain in storm water and bovine feces

Transfer of class 1 integron-mediated antibiotic resistance genes has been demonstrated under laboratory conditions. However, there is no information concerning the transfer of these genes in an agricultural environment. The present study sought to determine if integron-mediated streptomycin and sulfisoxazole resistance genes could be transferred from Shiga toxin-producing Escherichia coli (STEC) strains 6-20 (O157:H7) and 7-63 (O111:H8) to the susceptible strain E. coli K-12 MG1655 in bovine feces (pH 5.5, 6.0, or 6.5) and storm water (pH 5, 6, 7, or 8) at 4, 15, and 28 degrees C, which are average seasonal temperatures for winter, spring-fall, and summer, respectively, in the Griffin, GA, area. The results indicated that at 28 degrees C, the integron-mediated antibiotic resistance genes were transferred from both of the STEC donors in bovine feces. Higher conjugation efficiencies were, however, observed in the conjugation experiments involving STEC strain 6-20. In storm water, the resistance genes were transferred only from STEC strain 6-20. Greater numbers of transconjugants were recovered in the conjugation experiments performed with pH 6.5 bovine feces and with pH 7 storm water. Antibiotic susceptibility tests confirmed the transfer of integron-mediated streptomycin resistance and sulfisoxazole resistance, as well as the transfer of non-integron-mediated oxytetracycline resistance and tetracycline resistance in the transconjugant cells. These results suggest that the antibiotic resistance genes in STEC could serve as a source of antibiotic resistance genes disseminated via conjugation to susceptible cells of other E. coli strains in an agricultural environment.     

Human dehydroepiandrosterone sulfotransferase: purification and characterization of a recombinant protein

Dehydroepiandrosterone sulfate is the most abundant sulfated steroid transformed in human tissues and serves as a precursor for steroid hormones. Recombinant human dehydroepiandrosterone sulfotransferase (DHEA-ST) expressed in glutathione sulfotransferase fusion form in E. coli was purified using glutathione sepharose 4B affinity adsorption chromatography, a Factor Xa cleavage step, and Q-sepharose fast flow column chromatography. The homogeneous preparation had an activity toward dehydroepiandrosterone (DHEA) of 150+/-40 nmol/min per mg of protein under the assay conditions at an overall yield of 38.4%. The recombinant human DHEA-ST was shown to have a subunit mass of 34 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, while having a molecular mass of 67.2 kDa by Superose-12 gel filtration. Our results indicate that the active recombinant enzyme expressed in E. coli is a homodimer.Biochemical properties for purified DHEA-ST were studied using DHEA as a substrate. The optimum pH ranged from pH 7 to 8, and the optimum temperature 40-45 degrees C. Ninety percent of basal DHEA-ST activity remained even after the enzyme was treated at 45 degrees C for 15 min. The 50% inactivation concentration of NaCl for DHEA-ST activity was determined to be around 500 mM. The K(m) value for DHEA was 1.9+/-0.3 microM and V(max)=190+/-18 nmol/min per mg of protein at 37 degrees C, pH 7.5.     

HIV-1 protease: characterization of a catalytically competent enzyme-substrate intermediate.

The steady-state and pre-steady-state kinetic parameters for the interaction of E with the fluorogenic substrate 2-aminobenzoyl-Thr-Ile-Nle-Phe(p-NO(2))-Gln-Arg-NH(2) were determined in 1.25 M NaCl, 0.1 M MES-TRIS at pH 6.0 at 25 degrees C. At low concentrations of enzyme, the values of the K(m) and k(cat) calculated from steady-state data were 2.1 microM and 7.4 s(-1), respectively. At high concentrations of enzyme, the time-courses of fluorescence enhancement associated with catalysis were very dependent on the excitation wavelength used to monitor the reaction. Because the absorbance spectrum of the substrate overlapped the fluorescence emission spectrum of the enzyme, these abnormalities were attributed to fluorescence energy transfer between the enzyme and the substrate in an enzyme-substrate intermediate. The kinetic data collected with lambda(ex) = 280 nm and lambda(em) > 435 nm were analyzed according to the following mechanism in which EX was the species with enhanced fluorescence relative to substrate or products: [formula see text]. The values of the kinetic parameters with (1)H(2)O as the solvent were K = 13 microM, k(2) = 150 s(-1), k(-2) = 25 s(-1), and k(3) = 11 s(-1). The values of the kinetic parameters with (2)H(2)O as the solvent were K = 13 microM, k(2) = 210 s(-1), k(-2) = 12 s(-1), and k(3) = 4.4 s(-1). These values yielded solvent isotope effects of 2 on k(cat) and 0.9 on k(cat)/K(m). From analysis of the complete time-course of the fluorescence change (lambda(ex) = 280 nm and lambda(em) > 435 nm) during the course of substrate hydrolysis, the intermediate EX was determined to be 6.3-fold more fluorescent than the product, which, in turn, was 4.5-fold more fluorescent than ES or S. Rapid quench experiments with 2 N HCl as the quenching reagent confirmed that EX was a complex between enzyme and substrate. Consequently, the small burst in fluorescence observed when monitoring with lambda(ex) = 340 nm (0.3 product equiv per enzyme equivalent) was attributed to the fluorescence change upon transfer of substrate from an aqueous environment to a nonaqueous environment in the enzyme. These results were consistent with carbon-nitrogen bond cleavage being the major contributor to k(cat).     

Proton transfers in the beta-reaction catalyzed by tryptophan synthase

Reactions catalyzed by the beta-subunits of the tryptophan synthase alpha(2)beta(2) complex involve multiple covalent transformations facilitated by proton transfers between the coenzyme, the reacting substrates, and acid-base catalytic groups of the enzyme. However, the UV/Vis absorbance spectra of covalent intermediates formed between the pyridoxal 5'-phosphate coenzyme (PLP) and the reacting substrate are remarkably pH-independent. Furthermore, the alpha-aminoacrylate Schiff base intermediate, E(A-A), formed between L-Ser and enzyme-bound PLP has an unusual spectrum with lambda(max) = 350 nm and a shoulder extending to greater than 500 nm. Other PLP enzymes that form E(A-A) species exhibit intense bands with lambda(max) approximately 460-470 nm. To further investigate this unusual tryptophan synthase E(A-A) species, these studies examine the kinetics of H(+) release in the reaction of L-Ser with the enzyme using rapid kinetics and the H(+) indicator phenol red in solutions weakly buffered by substrate L-serine. This work establishes that the reaction of L-Ser with tryptophan synthase gives an H(+) release when the external aldimine of L-Ser, E(Aex(1)), is converted to E(A-A). This same H(+) release occurs in the reaction of L-Ser plus the indole analogue, aniline, in a step that is rate-determining for the appearance of E(Q)(Aniline). We propose that the kinetic and spectroscopic properties of the L-Ser reaction with tryptophan synthase reflect a mechanism wherein the kinetically detected proton release arises from conversion of an E(Aex(1)) species protonated at the Schiff base nitrogen to an E(A-A) species with a neutral Schiff base nitrogen. The mechanistic and conformational implications of this transformation are discussed.