Identification of essential histidine residues in the active site of Escherichia coli xylose (glucose) isomerase.
Two conserved histidine residues (His-101 and His-271) appear to be essential components in the active site of the enzyme xylose (glucose) isomerase (EC 5.3.1.5). These amino acid residues were targeted for mutagenesis on the basis of sequence homology among xylose isomerases isolated from Escherichia coli, Bacillus subtilis, Ampullariella sp. strain 3876, and Streptomyces violaceus-niger. Each residue was selectively replaced by site-directed mutagenesis and shown to be essential for activity. No measurable activity was observed for any mutations replacing either His-101 or His-271. Circular dichroism measurements revealed no significant change in the overall conformation of the mutant enzymes, and all formed dimers similar to the wild-type enzyme. Mutations at His-271 could be distinguished from those at His-101, since the former resulted in a thermolabile protein whereas no significant change in heat stability was observed for the latter. Based upon these results and structural data recently reported, we speculate that His-101 is the catalytic base mediating the reaction. Replacement of His-271 may render the enzyme thermolabile, since this residue appears to be a ligand for one of the metal ions in the active site of the enzyme.
Department of Food Science, Cornell University, Ithaca, NY 14853.
C A
CA
Batt
A C
AC
Jamieson
M A
MA
Vandeyar
D019747:Aldose-Ketose Isomerases; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002942:Circular Dichroism; D003062:Codon / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006639:Histidine; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid
D003062:Codon; D015345:Oligonucleotide Probes; D006639:Histidine; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase