GDP-manno
se hydrola
se catalyze
s the hydroly
si
s with inver
sion of GDP-
s/gifchar
s/alpha.gif" BORDER=0>-
D-hexo
se to GDP
and s/gifchar
s/beta2.gif" BORDER=0 ALIGN="middle">-
D-hexo
se by nucleophilic
sub
stitution by water at C1 of the
sugar. Two new cry
stal
structure
s (freeenzyme
and enzyme-
sub
strate complex), NMR,
and site-directed mutagene
si
s data, combined with the
structure of the enzyme-product complex reported earlier,
sugge
st a four-
stage catalytic cycle. An importantloop (L6, re
sidue
s 119-125) contain
s a lig
and to the e
ssential Mg
2+ (Gln-123), the catalytic ba
se (Hi
s-124),
and three anionic re
sidue
s. Thi
s loop i
s not ordered in the X-ray
structure of the free enzyme dueto dynamic di
sorder, a
s indicated by the two-dimen
sional
1H-
15N HMQC
spectrum, which
show
s selectiveexchange broadening of the imidazole nitrogen re
sonance
s of Hi
s-124 (
kex = 6.6 × 10
4 s-1). The
structureof the enzyme-Mg
2+-GDP-manno
se
sub
strate complex of the le
ss active Y103F mutant
show
s loop L6in an open conformation, while the
structure of the enzyme-Mg
2+-GDP product complex
showed loopL6 in a clo
sed, "active" conformation.
1H-
15N HMQC
spectra
show the imidazole N
s/gifchar
s/ep
silon.gif" BORDER=0 > of Hi
s-124 to beunprotonated, appropriate for general ba
se cataly
si
s. Sub
stituting Mg
2+ with the more electrophilic metalion
s Mn
2+ or Co
2+ decrea
se
s the p
Ka in the pH ver
su
s kcat rate profile
s,
showing that deprotonation of ametal-bound water i
s partially rate-limiting. The H124Q mutation, which decrea
se
s kcat 10
3.4-fold
andlargely aboli
she
s it
s pH dependence, i
s re
scued by the Y103F mutation, which increa
se
s kcat 23-fold
andre
store
s it
s pH dependence. The
structural ba
si
s of the re
scue i
s the fact that the Y103F mutation
shift
sthe conformational equilibrium to the open form moving loop L6 out of the active
site, thu
s permittingdirect acce
ss of the
specific ba
se hydroxide from the
solvent. In the propo
sed di
ssociative tran
sition
state,which occur
s in the clo
sed, active conformation of the enzyme, the partial negative charge of the GDPleaving group i
s compen
sated by the Mg
2+,
and by the clo
sing of loop L2 that bring
s Arg-37 clo
ser to the
s/gifchar
s/beta2.gif" BORDER=0 ALIGN="middle">-pho
sphate. The development of a po
sitive charge at manno
syl C1, a
s the oxocarbenium-like tran
sition
state i
s approached, i
s compen
sated by clo
sing the anionic loop, L6, onto the active
site, further
stabilizingthe tran
sition
state.