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.

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.

Identification of an active site histidine in urokinase

1976 ◽  
Vol 429 (1) ◽  
pp. 252-257 ◽  
Author(s):  
Eng Bee Ong ◽  
Alan J. Johnson ◽  
Guenther Schoellmann
Keyword(s):  
Active Site ◽  
Active Site Histidine

scholarly journals The location of the active-site histidine residue in the primary sequence of papain

1968 ◽  
Vol 108 (5) ◽  
pp. 861-866 ◽  
Author(s):  
S. S. Husain ◽  
G. Lowe
Keyword(s):  
Amino Acid ◽  
Sodium Borohydride ◽  
Active Site ◽  
Iodoacetic Acid ◽  
Cysteine Residue ◽  
Histidine Residue ◽  
Primary Sequence ◽  
Active Site Cysteine ◽  
Active Site Histidine ◽  
Active Site Cysteine Residue

Papain that had been irreversibly inhibited with 1,3-dibromo[2−14C]acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and α-chymotrypsin the radioactive peptides were purified chromatographically. Their amino acid composition indicated that cysteine-25 and histidine-106 were cross-linked. Since cysteine-25 is known to be the active-site cysteine residue, histidine-106 must be the active-site histidine residue.

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