Chemical Modification of Glucose Isomerase
Chemical modification of amino acid residues with specific
chemical reagents serves as a simple means of probing the
active site of the enzyme. The possible involvement of histidine
in the active site of GI was postulated by studying the effect of
diethylpyrocarbonate on the inactivation of GI (101). Later,
evidence for the presence of an essential histidine residue at
the active site of GI from different Lactobacillus spp. and
Streptomyces spp. (67, 171) was provided. Inhibition by diethylpyrocarbonate
was remedied by hydroxylamine. Total pro-
FIG. 2. Mechanism of action of GI. (a) cis-Enediol. (b) Proton transfer. (c) Hydride shift. Boxes indicate the hydrogen atoms that are transferred stereospecifically.
286 BHOSALE ET AL. MICROBIOL. REV.
tection of enzyme activity was afforded by the substrate and the
substrate analog xylitol during chemical modification. Histidine
is known to function as a proton-abstracting base and to
assist in hydrogen transfers (Fig. 2b). The presence of an aspartate
or glutamate residue in GI was documented by its
inactivation by Woodward’s reagent K or guanidine hydrochloride
(74, 172). Involvement of carboxylate residues is implicated
in the binding of metal ion cofactors (34). Chemical
modification of protected and unprotected GI and subsequent
peptide mapping allowed the identification of an active-site
region with a consensus sequence consisting of Phe-His-Xaa-
Asp-Xaa-Xaa-Pro-Xaa-Gly (173). The results of studies on the
chemical modification of GI complement the conclusions
drawn on the basis of X-ray crystallographic studies.