| dc.description.abstract | Uronate isomerase catalyzes the isomerization of D-glucuronate and Dfructuronate.
This enzyme has been classified as a member of the amidohydrolase
superfamily. The reaction catalyzed by uronate isomerase is analogous to the
isomerization of aldose/ketose sugars. These interconversions can occur via two
mechanisms, a hydride or proton transfer. The solvent exchange experiments and the
elimination of fluoride from 3-deoxy-3-fluoro-D-glucuronate catalyzed by the enzyme
support a proton transfer. Assignment of the transferred proton as the proR proton further
supports a proton transfer mechanism via a cis-enediol intermediate for uronate
isomerase from E. coli.
Cobyrinic acid a,c-diamide synthetase and cobyric acid synthetase from S.
typhimurium catalyze ATP dependent amidations of carboxylate groups on the periphery
of cobyrinic acid utilizing glutamine or ammonia as a nitrogen donor. The role of ATP in
the reaction has been probed by positional isotope exchange (PIX). The results confirm the presence of phosphorylated intermediate species in the reactions catalyzed by
cobyrinic acid a,c-diamide synthetase and cobyric acid synthetase from S. typhimurium.
Cobyric acid synthetase catalyzes the amidation of carboxylate groups b, d, e, and
g of adenosyl-cobyrinic acid a,c-diamide in the biosynthetic pathway for coenzyme B12.
Analysis of the reaction time courses demonstrate the appearance of three unique
intermediate species which are released from the active site after each amidation
reaction. The identification of the intermediate species was accomplished by 1H, 15N
HSQC NMR spectroscopy. The NMR spectrum of a sample quenched at the beginning
of the reaction shows a single intermediate species corresponding to carboxamide e.
Subsequent spectra establish the amidation order as e, d, b, and g. The structural basis
for the dissociative and sequential reaction mechanism coupled with the rigid
regiochemistry is unknown. However, mutations to aspartate 146 perturb the order of
amidation. A NMR spectrum quenched early in the reaction with the D146N mutant
shows two intermediate species corresponding to carboxamides e and d. Spectra of
samples later in the reaction confirm the presence of multiple e, d, and g amide species.
The reaction is completed with the amidation of carboxylate b. | en |
