Journal of the American Chemical Society
COMMUNICATION
Scheme 4. Deduced Conformation of the Transition State
Leading to the Observed KIE (the Enzyme Active Site Is
Represented by an Incomplete Border)
compound synthesis, and characterization; and kinetics and enzy-
matic reaction conditions. This material is available free of charge via
’ AUTHOR INFORMATION
Corresponding Author
’ ACKNOWLEDGMENT
We thank the Natural Sciences and Engineering Research
Council of Canada, the Canada Research Chairs Program, the
Canadian Foundation for Innovation, and the B.C. Knowledge
Development Fund for financial support.
the pair of substrates 7 and 8, having either hydrogen or deuterium
at carbon 4, were synthesized as described in the SI. The trichlor-
ophenyl leaving group was used here to allow ease of synthetic
access to the product through hydrogenation. The KIE on kcat/KM
was measured spectrophotometrically in individual reactions to be
1.06 ( 0.02 and confirmed by direct competition in one reaction
monitored by 1H NMR analysis (1.08 ( 0.03). This small effect was
initially surprising, as an inverse α-secondary KIE had been antici-
pated. However, previous measurements of KIEs for hydration of
CÀC double bonds under acidic conditions have revealed values
around unity.16,17 This low value is attributed to equal but opposite
effects from a change in hybridization from sp2 to sp3 at the site of
substitution (giving an inverse α-secondary effect) and hyperconju-
gation with the adjacent carbocation at carbon 5 (giving a normal
β-secondary effect). Since such β-secondary KIEs are highly depen-
dent on geometry because of the need for alignment of the CÀD
bond and the adjacent empty p orbital, the finding of a net KIE of
near unity implies a relatively large β-secondary effect to balance the
expected inverse α-secondary effect. In turn, this suggests a transi-
tion-state geometry such as that shown in Scheme 4, with the CÀD
bond axial.
In summary, evidence has been provided here that clearly
demonstrates the use of a novel hydration mechanism by unsatu-
rated glucuronyl hydrolases to effect cleavage of glycosidic bonds:
the proton addition at carbon 4 has been shown to occur in a
stereospecific manner, and nucleophilic attack has been shown to
occur from the same face at carbon 5. The reaction directly catalyzed
is the syn hydration of a vinyl ether to give an unstable hemiketal,
and it is the collapse of this species that leads to glycosidic bond
cleavage. The present data do not allow us to determine whether this
collapse occurs on the enzyme or free in solution. The formation of a
glycosylÀenzyme intermediate through syn addition of the general
acid catalyst residue was ruled out, in contrast to the established
mechanism for enzymatic hydration of glycals. A secondary deuter-
ium kinetic isotope effect on kcat/KM from a 4-deuterated substrate
was observed, also consistent with the proposed mechanism invol-
ving an oxocarbenium ion-like transition state, indicating that this
deuterium adopts an axial geometry in the transition state. The
leaving group was seen to be unimportant in the catalytic mechan-
ism, being important only in binding. In fact, in the case where it was
replaced by a carbon-linked glycerol chain, the enzyme could still
catalyze the hydration reaction. Confirmation of this mechanism
therefore further expands the body of known enzymatic strategies
employed for cleavage of glycosides.
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’ ASSOCIATED CONTENT
1
S
Supporting Information. Overlay of H NMR spectra
b
for the 10% methanol product with all controls and standards;
COSY and NOESY spectra; details of cloning, enzyme purification,
19337
dx.doi.org/10.1021/ja209067v |J. Am. Chem. Soc. 2011, 133, 19334–19337