C O M M U N I C A T I O N S
Scheme 3
Table 1. Kinetic Parameters Extracted for IMPase Enzymes
A. fulgidus IMPase (85
m (mM)
°
C)
E. coli SuhB (37
m (mM)
°
C)
substratea
K
k
cat (s-
)
K
k
cat(s-
)
1
1
D-I-1P (1)
3-deoxy (8)
3,5-dideoxy (11)
L-I-1P (D-I-3P, ent-1)
3-deoxy (ent-8)
0.11 ( 0.02
0.23 ( 0.05
0.16 ( 0.01
1.7 ( 0.4
3.3 ( 0.2
3.9 ( 0.3
2.5 ( 0.1
3.6 ( 0.2
3.2 ( 0.3
5.8 ( 0.4
0.068 ( 0.01
0.047 ( 0.005
0.051 ( 0.008
0.061 ( 0.03
0.20 ( 0.03
4.4 ( 0.3
4.4 ( 0.1
4.0 ( 0.2
1.4 ( 0.2
5.6 ( 0.4
4.4 ( 0.3
2.3 ( 0.6
3,5-dideoxy (ent-11)
4.9 ( 0.8
0.10 ( 0.02
a Assays were carried out in 50 mM Tris HCl, pH 8.0, with 4 and 8 mM
free Mg2+ for the archaeal and E. coli enzymes, respectively.
Whatever the rate-limiting step, the activation energy for ent-1
hydrolysis must be reduced when the C1-hydroxyl group of ent-1
is removed. Since no crystal structure exists of the E. coli IMPase,
we can predict that in terms of substrate binding, the interactions
of the enzyme with the C3-OH group of L-I-1P may be destabiliz-
ing. Thus, the substrate kinetics with SuhB are reminiscent of those
observed with the mammalian IMPase,2b whereas the archaeal
system exhibits very different behavior.
Can the kinetics with these defined inositol phosphate isomers
contribute to our understanding of the biology of the archaeal
enzyme? In A. fulgidus, the IMPase is involved in synthesizing an
unusual solute (L,L′-di-myo-inositol-1,1′-phosphate, DIP),8 which
is used for osmotic balance but also produced in response to heat
stress.9 Biosynthesis of DIP requires both L-I-1P (converted to CDP-
inositol) and myo-inositol (produced from L-I-1P by the IMPase).
To ensure that there is adequate L-I-1P for DIP synthesis, the IMPase
needs to be regulated. The dramatically increased Km for L-I-1P
could well be part of this control since little myo-inositol would be
generated until a significant amount of L-I-1P accumulated.
In summary, rapid synthesis of point-by-point “mutated” IPs can
shed light on their interactions with protein targets, and uncover
differences among proteins. Expanded studies of these compounds,
and their phosphatidyl analogues, should increase our understanding
of their roles in biology.
stereochemically pure form (99% yield). With enantiodivergent
catalysts 3 and 4 in hand for the asymmetric phosphorylation, we
also carried out the identical sequence in the D-myo-inositol-3-phos-
phate series to give ent-8 (1-deoxy-D-myo-inositol-3-phosphate6).
The preparation of the 3,5-dideoxy-compounds 11 and ent-11
followed an analogous plan (Scheme 3). In this case, both the 3-
and 5-positions may be subjected to thiocarbonylation for prolonged
reaction time (due to the lower reactivity of the 5-hydroxyl group.
Thus, bis(thiocarbonate)-9 could be obtained in 70% yield. Simul-
taneous radical deoxygenation, followed by dissolving metal
reduction to cleave the protecting groups, affords 11 (61% yield).
The D-I-1P series (1, 8, 11) and the L-I-1P series (D-I-3P series:
ent-1, ent-8, ent-11) with 3-deoxy and 3,5-dideoxy alterations were
examined as substrates for A. fulgidus IMPase (assayed at 85 °C)
and for E. coli SuhB (assayed at 37 °C). The latter enzyme has
many kinetics characteristics similar to those of eukaryotic IMPase
enzymes.7 The A. fulgidus IMPase crystal structure with D-I-1P
bound shows hydrogen-bond interactions between the inositol C3-
hydroxyl and Tyr155 (polarized by Glu175) and the amide nitrogen
of Ala172. Consistent with this, the Km for 8 is increased compared
to 1. The ∆∆G extracted from this increase in Km reflects a change
of only ∼2.2 kJ/mol. Removal of the C-5 hydroxyl group did not
significantly increase Km further. More interesting was the very
pronounced increase in Km for all the D-I-3P or (L-series) substrates
for the archaeal IMPase (Table 1). The averaged ∆∆G reflected in
the increased Km of A. fulgidus IMPase for a specific L- versus D-
substrate was 8.4 ( 1.0 kJ/mol. A comparison of Km within the
L-I-1P (D-I-3P) series, showed much smaller increases: the differ-
ence in Km for ent-8 versus ent-1 reflects only about 1 kJ/mol, while
removing both 3- and 5-hydroxyl groups (ent-11) in this series costs
about 3 kJ/mol for formation of the Michaelis complex. The results
suggest that when L-I-1P is bound in the active site, the C5-hydroxyl
group is likely to interact with the protein, while the C3-hydroxyl
group has a considerably weaker interaction. More critically, the
large difference in Km between the L- and D- series is equivalent to
removing a hydrogen-bonding interaction of the L-I-1P ligand with
the protein. [For comparison, the mammalian IMPase binds D- and
L-I-1P somewhat differently (60° change in orientation), but without
significantly affecting kinetic parameters.2c] Removal of hydroxyl
groups also affected the kcat of the A. fulgidus IMPase differently
for the D-I-1P versus L-I-1P series. Removal of the two hydroxyl
groups decreased kcat with the D-I-1P series but increased kcat for
the L-I-1P series.
Acknowledgment. This work is supported by the NIH (NIGMS-
68649 to S.J.M. and DE-FG02-91ER20025 to M.F.R). We also
acknowledge Pfizer for support. A.J.M. is grateful to Organic
Syntheses and the ACS (ORGN) for a fellowship.
Supporting Information Available: Experimental details and
characterization for the synthetic chemistry and the enzyme assay. This
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The results for SuhB provide an intriguing contrast. The D-I-1P
series showed no significant change in Km as hydroxyl groups were
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