Evidence for an essential histidine residue in D-xylose isomerases.
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.
Vangrysperre
W
W
Laboratorium voor Biochemie, Rijksuniversiteit Gent, Belgium.
Callens
M
M
Kersters-Hilderson
H
H
De Bruyne
C K
CK
D019747:Aldose-Ketose Isomerases; D064753:Ammonium Hydroxide; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine / Q000378:metabolism*; D006878:Hydroxides / Q000494:pharmacology; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology; D008024:Ligands; D013053:Spectrophotometry; D013291:Streptococcus / Q000201:enzymology
D002413:Cations, Divalent; D006878:Hydroxides; D008024:Ligands; D006639:Histidine; D064753:Ammonium Hydroxide; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D004047:Diethyl Pyrocarbonate