Phosphoglucose isomerase (EC 5.3.1.9) catalyzes
the interconversion of d-glucopyranose-6-phosphate
and d-fructofuranose-6-phosphate by promoting
an intrahydrogen transfer between C1 and C2. A conserved
histidine exists throughout all phosphoglucose isomerases
and was hypothesized to be the base catalyzing the isomerization
reaction. In the present study, this conserved histidine,
His311, of the enzyme from Bacillus stearothermophilus
was subjected to mutational analysis, and the mutational
effect on the inactivation kinetics by N-bromoacetylethanolamine
phosphate was investigated. The substitution of His311
with alanine, asparagine, or glutamine resulted in the
decrease of activity, in
kcat/KM,
by a factor of 103, indicating the importance
of this residue. N-bromoacetylethanolamine phosphate inactivated
irreversibly the activity of wild-type phosphoglucose isomerase;
however, His311 → Ala became resistant to this inhibitor,
indicating that His311 is located in the active site and
is responsible for the inactivation of the enzyme by this
active site-directed inhibitor. The pKa
of His311 was estimated to be 6.31 according to the pH
dependence of the inactivation. The proximity of this value
with the pKa value of 6.35, determined
from the pH dependence of
kcat/KM,
supports a role of His311 as a general base in the catalysis.