scholarly journals Evidence for an essential histidine residue in d-xylose isomerases

1988 ◽  
Vol 250 (1) ◽  
pp. 153-160 ◽  
Author(s):  
W Vangrysperre ◽  
M Callens ◽  
H Kersters-Hilderson ◽  
C K De Bruyne
Keyword(s):  
Xylose Isomerase ◽  
Lactobacillus Brevis ◽  
Histidine Residue ◽  
Difference Spectra ◽  
Streptomyces Sp ◽  
Histidine Residues ◽  
Tyrosine Residues ◽  
Diethyl Pyrocarbonate ◽  
The Difference ◽  
Streptomyces Violaceoruber

Diethyl pyrocarbonate inactivated D-xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus and Lactobacillus brevis with second-order rate constants of 422, 417, 99 and 92 M-1.min-1 respectively (at pH 6.0 and 25 degrees C). Activity was completely restored by the addition of neutral hydroxylamine, and total protection was afforded by the substrate analogue xylitol in the presence of either Mg2+ or Mn2+ according to the genus studied. The difference spectra of the modified enzymes revealed an absorption maximum at 237-242 nm, characteristic for N-ethoxycarbonylhistidine. In addition, the spectrum of ethoxycarbonylated D-xylose isomerase from L. xylosus showed absorption minima at both 280 and 230 nm, indicative for modification of tyrosine residues. Nitration with tetranitromethane followed by diethyl pyrocarbonate treatment eliminated the possibility that modification of tyrosine residues was responsible for inactivation, and resulted in modification of one non-essential tyrosine residue and six histidine residues. Inactivation of the other D-xylose isomerases with diethyl pyrocarbonate required the modification of one (L. brevis), two (Streptomyces sp.) and four (S. violaceoruber) histidine residues per monomer. Spectral analysis and maintenance of total enzyme activities further indicated that either xylitol Mg2+ (streptomycetes) or xylitol Mn2+ (lactobacilli) prevented the modification of one crucial histidine residue. The overall results thus provide evidence that a single active-site histidine residue is involved in the catalytic reaction mechanism of D-xylose isomerases.

scholarly journals Modification of lactate oxidase with diethyl pyrocarbonate. Evidence for an active-site histidine residue

1977 ◽  
Vol 165 (2) ◽  
pp. 385-393 ◽  
Author(s):  
Choong Yee Soon ◽  
Maxwell G. Shepherd ◽  
Patrick A. Sullivan
Keyword(s):  
Enzyme Activity ◽  
Chemical Reduction ◽  
Enzyme Inactivation ◽  
Subunit Structure ◽  
Histidine Residue ◽  
Lactate Oxidase ◽  
Histidine Residues ◽  
Diethyl Pyrocarbonate ◽  
Competitive Inhibitors ◽  
Active Site Histidine

1. Diethyl pyrocarbonate inactivated l-lactate oxidase from Mycobacterium smegmatis. 2. Two histidine residues underwent ethoxycarbonylation when the enzyme was treated with sufficient reagent to abolish more than 90% of the enzyme activity, but analyses of the inactivation showed that the modification of one histidine residue was sufficient to cause the loss of enzyme activity. The rates of enzyme inactivation and histidine modification were the same. 3. Substrate and competitive inhibitors decreased the maximum extent of inactivation to a 50% loss of enzyme activity and modification was decreased from 1.9 to 0.75–1.2 histidine residues modified/molecule of FMN. 4. Treatment of the enzyme with diethyl [14C]pyrocarbonate (labelled in the carbonyl groups) confirmed that only histidine residues were modified under the conditions used and that deacylation of the ethoxycarbonylhistidine residues by hydroxylamine was concomitant with the removal of the14C label and the re-activation of the enzyme. 5. No evidence was found for modification of tryptophan, tyrosine or cysteine residues, and no difference was detected between the conformation and subunit structure of the modified and native enzyme. 6. Modification of the enzyme with diethyl pyrocarbonate did not alter the following properties: the binding of competitive inhibitors, bisulphite and substrate or the chemical reduction of the flavin group to the semiquinone or fully reduced states. The normal reduction of the flavin by lactate was, however, abolished.

scholarly journals The histidine residues of phospholipase C from Bacillus cereus

1977 ◽  
Vol 167 (2) ◽  
pp. 399-404 ◽  
Author(s):  
C Little
Keyword(s):  
Catalytic Activity ◽  
Bacillus Cereus ◽  
Phospholipase C ◽  
Maximum Velocity ◽  
Native Enzyme ◽  
Histidine Residue ◽  
Histidine Residues ◽  
Residue Removal ◽  
Diethyl Pyrocarbonate ◽  
Km Value

The inactivation of phospholipase C from Bacillus cereus at pH6 by diethyl pyrocarbonate parallelled the N-ethoxyformylation of a single histidine residue in the enzyme. The inactivation arose from a decrease in the maximum velocity of the enzymic reaction with no effect on the Km value. The inactivation did not apparently alter the ability of the enzyme to bind to a substrate-based affinity gel. The native enzyme contained only one reactive histidine residue. Removal of the two zinc atoms from the enzyme increased the number of reactive histidine residues to five, whereas in the totally denatured enzyme nearly eight such residues were available for reaction with diethyl pyrocarbonate. The enzyme thus appears to contain one histidine residue that is essential for catalytic activity and four that may be involved in co-ordinating the zinc atoms in the structure.

scholarly journals Modification of uridine phosphorylase from Escherichia coli by diethyl pyrocarbonate. Evidence for a histidine residue in the active site of the enzyme

1990 ◽  
Vol 270 (2) ◽  
pp. 319-323 ◽  
Author(s):  
A K Drabikowska ◽  
G Woźniak
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Active Site ◽  
Order Rate Constant ◽  
Enzymic Activity ◽  
Histidine Residue ◽  
Uridine Phosphorylase ◽  
High Concentration ◽  
Histidine Residues ◽  
Diethyl Pyrocarbonate

Uridine phosphorylase from Escherichia coli is inactivated by diethyl pyrocarbonate at pH 7.1 and 10 degrees C with a second-order rate constant of 840 M-1.min-1. The rate of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.6. Hydroxylamine added to the inactivated enzyme restores the activity. Three histidine residues per enzyme subunit are modified by diethyl pyrocarbonate. Kinetic and statistical analyses of the residual enzymic activity, as well as the number of modified histidine residues, indicate that, among the three modifiable residues, only one is essential for enzyme activity. The reactivity of this histidine residue exceeded 10-fold the reactivity of the other two residues. Uridine, though at high concentration, protects the enzyme against inactivation and the very reactive histidine residue against modification. Thus it may be concluded that uridine phosphorylase contains only one histidine residue in each of its six subunits that is essential for enzyme activity.

scholarly journals Reaction of Woodward's reagent K with d-xylose isomerases. Modification of an active site carboxylate residue

1989 ◽  
Vol 260 (1) ◽  
pp. 163-169 ◽  
Author(s):  
W Vangrysperre ◽  
H Kersters-Hilderson ◽  
M Callens ◽  
C K De Bruyne
Keyword(s):  
Spectral Analysis ◽  
Catalytic Activity ◽  
Active Site ◽  
Rate Constants ◽  
Bacillus Coagulans ◽  
Lactobacillus Brevis ◽  
Carboxylate Group ◽  
Streptomyces Sp ◽  
Woodward’S Reagent K ◽  
Streptomyces Violaceoruber

D-Xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus, Lactobacillus brevis and Bacillus coagulans were rapidly inactivated by Woodward's reagent K. Second-order rate constants in the absence of ligands, at pH 6.0 and 25 degrees C, were 41, 36, 22, 95 and 26 M-1.min-1 respectively. Spectral analysis at 340 nm revealed that inactivation was correlated with modification of five, six, two, three and six carboxylate residues per monomer respectively. In the presence of protecting ligands, modification of one carboxylate group was prevented. The results support the idea of an active site glutamate or aspartate group that may contribute to the catalytic activity of all these D-xylose isomerases.

Photoreaction of tyrosin-iodinated bacteriorhodopsin at low temperature

1982 ◽  
Vol 2 (11) ◽  
pp. 949-958 ◽  
Author(s):  
Tatsuo Iwasa ◽  
Kazuo Takeda ◽  
Fumio Tokunaga ◽  
Peter S. Scherrer ◽  
Lester Packer
Keyword(s):  
Low Temperature ◽  
Proton Pump ◽  
Photochemical Reaction ◽  
Room Temperature ◽  
Green Light ◽  
Glycerol Solution ◽  
Difference Spectra ◽  
Tyrosine Residues ◽  
The Difference

To elucidate the role of tyrosine residues in the shift of λmax and the light-driven proton pump of bacteriorhodopsin, the photochemical reaction of tyrosine-iodinated bacteriorhodopsin (tyr-mod-bR) was investigated by low-temperature spectrophotometry. After 4–5 of 11 tyrosine residues of bacteriorhodopsin were iodinated, the meta-intermediate of tyr-mod-bR in 75% glycerol solution became so stable that its decay could be observed even at room temperature and it was stable in the dark for several hours at −65°C. Four batho-intermediates were formed by irradiation with green light (500 nm) at −170°C. Like native bacteriorhodopsin, these batho-intermediates were photoreversible at −170°C. Four corresponding meta-intermediates were also formed by irradiation at −60°C. Using the difference spectra between meta-intermediates and tyr-mod-bR, the absorption spectra of four kinds of tyr-mod-bRs, batho-intermediates, and meta-intermediates were estimated. Each was at shorter wavelengths than that of its corresponding type in native bacteriorhodopsin. The results indicate that two or more tyrosine residues have some role in determining color in native bacteriorhodopsin.

scholarly journals Single active-site histidine in d-xylose isomerase from Streptomyces violaceoruber. Identification by chemical derivatization and peptide mapping

1989 ◽  
Vol 263 (1) ◽  
pp. 195-199 ◽  
Author(s):  
W Vangrysperre ◽  
C Ampe ◽  
H Kersters-Hilderson ◽  
P Tempst
Keyword(s):  
Active Site ◽  
Peptide Mapping ◽  
Xylose Isomerase ◽  
Specific Chemical ◽  
Peptide Cleavage ◽  
Diethyl Pyrocarbonate ◽  
Streptomyces Violaceoruber ◽  
Wavelength Detection ◽  
Micro Organisms ◽  
Active Site Histidine

Group-specific chemical modifications of D-xylose isomerase from Streptomyces violaceruber indicated that complete loss of activity is fully correlated with the acylation of a single histidine. Active-site protection, by the ligand combination of xylitol plus Mg2+, completely blocked diethyl pyrocarbonate derivatization of this particular residue [Vangrysperre, Callens, Kersters-Hilderson & De Bruyne (1988) Biochem. J. 250, 153-160]. Differential peptide mapping between D-xylose isomerase, which has previously been treated with diethyl pyrocarbonate in the presence or absence of xylitol plus Mg2+, allowed specific isolation and sequencing of a peptide containing this active-site histidine. For this purpose we used two essentially new techniques: first, a highly reproducible peptide cleavage protocol for protease-resistant, carbethoxylated proteins with guanidinium hydrochloride as denaturing agent and subtilisin for proteolysis; and second, reverse-phase liquid chromatography with dual-wavelength detection at 214 and 238 nm, and calculation of absorbance ratios. It allowed us to locate the single active-site histidine at position 54 in the primary structure of Streptomyces violaceoruber D-xylose isomerase. The sequence around this residue is conserved in D-xylose isomerases from a diversity of micro-organisms, suggesting that this is a structurally and/or functionally essential part of the molecule.

The interaction of bovine milk caseins with the detergent sodium dodecyl sulphate. II. The effect of detergent binding on spectral properties of caseins

1970 ◽  
Vol 37 (2) ◽  
pp. 259-267 ◽  
Author(s):  
G. C. Cheeseman ◽  
Dorothy J. Knight
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Bovine Milk ◽  
Difference Spectrum ◽  
Temperature Dependent ◽  
Difference Spectra ◽  
Negative Change ◽  
Tryptophan Residues ◽  
Hydrophobic Binding ◽  
The Difference

SummaryThe dissociation of casein aggregates by the detergent sodium dodecyl sulphate (SDS) gave rise to difference spectra and these spectra were characteristic for each of the different types of casein. Increase in absorption by the chromophore groups, tyrosine and tryptophan, when αs1- and β-casein aggregates were dissociated indicated binding of the detergent at regions of the molecule containing these residues. A decrease in absorption when κ-casein was dissociated indicated that the tyrosine and tryptophan residues were not in the region of the molecule to which the detergent was bound and that in the κ-casein aggregate these residues were in a more hydrophobic environment. Peaks on the difference spectra were obtained at 280 and 288 nm for αs1-casein and 284 and 291 nm for β-casein and troughs at 278 and 286 nm for κ-casein. The difference spectrum reached a maximum value when the αsl- and β-casein aggregates were dissociated and the further binding of SDS did not alter this value. The large negative change in the difference spectrum of κ-casein did not occur until after most of the aggregates were dissociated and did not reach a maximum until binding with SDS was complete. The value obtained for ΔOD was found to be temperature-dependent for β-casein-SDS interaction, but not for αs1- and κ-casein. Changes in spectra were also observed when αs1- and κ-casein interacted to form aggregates. The data obtained confirmed the importance of hydrophobic binding in casein aggregate formation and indicated the possible involvement of tyrosine and tryptophan residues in this binding.

scholarly journals Assessment of Conformational Changes in Low-Sulphur S-Carboxymethylkerateine from Wool

1967 ◽  
Vol 20 (4) ◽  
pp. 827 ◽  
Author(s):  
GM Bhatnagar ◽  
WG Crewther
Keyword(s):  
Absorption Spectrum ◽  
Conformational Changes ◽  
Guanidine Hydrochloride ◽  
Ultraviolet Absorption Spectrum ◽  
Spectral Measurements ◽  
Characteristic Difference ◽  
Concentrated Solutions ◽  
Difference Spectra ◽  
The Difference ◽  
Negative Difference

The effects of urea and guanidine hydrochloride on the ultraviolet absorption spectrum of the low-sulphur S-carboxymethylkerateine fraction of wool have been measured. In concentrated solutions of urea characteristic difference spectra were obtained with maxima of negative absorbance at 288, 280, and 240 miL. The addition of guanidine hydrochloride or an increase in temperature gave similar negative difference maxima at the higher wavelengths. Calculation of the extent of unfolding of the protein chains from the difference in absorbance at all three maxima showed that the unfolding was 50% complete at a urea concentration of about 1� 8M whereas a urea concep.tration of about 4� 3M was required to decrease the helix content by 50%. Similar measurements on components 7 and 8, the two major constituents of SCMKA, showed that a 50% decrease in helix content was obtained with 2�8M and O� 8M urea respectively whereas the corresponding values for 50 % unfolding assessed from difference spectral measurements were 2� 2M and 1� 2M urea respectively. It is suggested that the helical regions of components 7 and 8 aggregate specifically and that spectral measurements relate largely to non-helical portions of the chains.

Unusually Strong Bi-Exciton Repulsion Detected in Quantum Confined CsPbBr3 Nanocrystals with Two and Three Pulse Femtosecond Spectroscopy

2021 ◽  
Author(s):  
jayanta dana ◽  
Tal Binyamin ◽  
Lioz Etgar ◽  
Sanford Ruhman
Keyword(s):  
Cross Section ◽  
Quantum Confinement ◽  
Auger Recombination ◽  
Transient Absorption ◽  
Stimulated Emission ◽  
Band Edge ◽  
High Time Resolution ◽  
Difference Spectra ◽  
The Difference ◽  
Absorption Measurements

Transient absorption measurements were conducted on pristine and on monoexciton saturated<br>CsPbBr3 nanocrystals varying in size within the regime of strong quantum confinement. Once<br>the difference spectra were translated to absolute transient changes in absorption cross section,<br>a single exciton is shown to completely bleach the band edge absorption peak, and induce a<br>new absorption roughly two times weaker ~100 meV to the blue. Difference spectra obtained<br>during Auger recombination of biexcitons demonstrate that addition of a second exciton, rather<br>than double the effect of a first, bleaches the blue induced absorption band without producing<br>net stimulated emission at the band edge. Accompanied by high time resolution transient<br>absorption spectra pumping at the lowest exciton band, these results identify the blue induced<br>absorption as the second transition to 1Se1Sh which is shifted in energy due to unusually strong<br>and promptly rising biexciton repulsion. <br>

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