Single active-site histidine in D-xylose isomerase from Streptomyces violaceoruber. Identification by chemical derivatization and peptide mapping.
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.
Vangrysperre
W
W
Laboratory of Biochemistry, Ghent State University, Belgium.
Ampe
C
C
Kersters-Hilderson
H
H
Tempst
P
P
D019747:Aldose-Ketose Isomerases; D000595:Amino Acid Sequence; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D006639:Histidine / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D012689:Sequence Homology, Nucleic Acid; D013056:Spectrophotometry, Ultraviolet; D013302:Streptomyces / Q000201:enzymology*
D006639:Histidine; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase