Inversion of configuration during the hydrolysis of D-1-S(p)-myo- inositol [17O]thiophosphate catalyzed by myo-inositol monophosphatase

Full text of DOI:10.1021/ja991939r

J. Am. Chem. Soc. 1999, 121, 8385-8386
8385
Communications to the Editor
reaction.7 For enzyme-catalyzed phosphoryl-transfer reactions that
occur with overall retention of configuration, there is usually
strong independent evidence for a double displacement involving
a phosphoenzyme intermediate.7 In the case of myo-inositol
monophosphatase, a phosphoenzyme appears to be ruled out on
the basis of the detailed kinetic analysis and by virtue of the failure
to identify an appropriately positioned enzyme nucleophile in the
X-ray crystal structure. However, retention of configuration would
also be observed for a mechanism involving direct phosphoryl
transfer via a single pseudorotation step.7a Thus, we report here
the stereochemical course of the hydrolysis of D-1-Sp-myo-inositol
[17O]thiophosphate catalyzed by recombinant bovine myo-inositol
monophosphatase.
The methodology for the determination of the stereochemical
course of enzyme-catalyzed phosphoryl-transfer reaction is well
established.7 For reactions catalyzed by phosphatases, it is
necessary to resort to thiophosphate analogues in order to end up
with a chiral product, viz., inorganic [16O,17O,18O]thiophosphate.
The synthesis of D-1-Sp-myo-inositol [17O]thiophosphate is shown
in Scheme 1.8 It has already been established that myo-inositol
monophosphatase catalyzes the hydrolysis of thiophosphate esters,
albeit at reduced rates compared to the corresponding phosphate
esters.6d,9 In terms of the practicalities of determining the
stereochemical course of the reaction, it is essential to optimize
the conditions to allow accumulation of inorganic thiophosphate
and to minimize the competing loss of sulfur that follows the
enzymatic step. Hydrolysis of D-1-Sp-myo-inositol [17O]-thiophos-
phate (90 µmol, added portionwise every 2 h over 32 h to
minimize the substrate concentration to avoid substrate inhibition)
in [18O]water (4 mL, buffered with Tris-HCl at pH 9.0), catalyzed
by recombinant bovine myo-inositol monophosphatase10 (31 mg,
previously lyophilised with [18O]water), led to the isolation of
inorganic [16O,17O,18O]thiophosphate (ca. 20 µmol) of unknown
configuration. The configurational analysis of this material was
achieved by the chemical method developed by Lowe et al.11
Inversion of Configuration during the Hydrolysis of
D-1-Sp-myo-Inositol [17O]Thiophosphate Catalyzed by
myo-Inositol Monophosphatase
Christine M. J. Fauroux,‡ Michael Lee,† Paul M. Cullis,*,†
Kenneth T. Douglas,‡ Sally Freeman,*,‡ and Michael G. Gore§
Department of Chemistry, The UniVersity of Leicester
Leicester LE1 7RH, U.K.
School of Pharmacy and Pharmaceutical Sciences
Oxford Road, Manchester UniVersity
Manchester M13 9PL, U.K.
DiVision of Biochemistry and Molecular Biology
School of Biological Sciences, UniVersity of
Southampton, Southampton SO16 7PX, U.K.
ReceiVed June 10, 1999
Inositol monophosphatase (EC 3.1.3.25) catalyzes the hydroly-
sis of D-myo-inositol 1-, 3-, 4-, 5-, and 6-monophosphates.1 This
enzyme has attracted considerable attention because it plays a
crucial role in the regulation of myo-inositol for the phosphatidyl
inositol cell signaling pathway.2 This pathway is thought to be
overactivated in patients with manic depression, and inositol
monophosphatase may be the in vivo target for its treatment with
lithium.3 The brain enzymes from a number of mammalian sources
are very similar, comprising a homodimer of subunit Mr ≈ 30
kDa, and the cloned human brain enzyme has been crystallized
and the X-ray structure determined.4 The structure of a catalytic-
ally inactive ternary (enzyme-substrate-metal) complex showed
the active site to contain two metal ions, and the involvement of
two metals in enzyme turnover has been confirmed by extensive
kinetic investigation.5,6 Notwithstanding this detailed structural
and kinetic investigation, the fundamental details of the mechan-
ism of this important enzyme remain obscure. Arguments based
on these kinetic studies have been advanced to reject an in-line
mechanism in favor of this enzyme catalyzing the hydrolysis of
myo-inositol monophosphate via an adjacent attack involving a
pseudorotation step.6
(7) (a) Knowles, J. R. Annu. ReV. Biochem. 1980, 49, 877-919. (b)
Buchwald, S. L.; Hansen, D. E.; Hassett, A.; Knowles, J. R. Methods Enzymol.
1982, 87, 279-301. (c) Eckstein, F. Angew. Chem., Int. Ed. Engl. 1983, 22,
423. (d) Frey, P. A. Tetrahedron 1982, 38, 1541. (e) Lowe, G. Acc. Chem.
Res. 1983, 16, 244. (f) Frey, P. A. AdV. Enzymol. Relat. Areas Mol. Biol.
1989, 62, 119.
(8) (D/L)-1,2;4,5-Di-O-cyclohexylidene-myo-inositol was synthesized ac-
cording to literature methods: Dreef, C. E.; Tulnman, R. J.; Lefeber, A. W.
M. Tetrahedron 1991, 47, 4709. The assignment of the diastereomers was
made by comparison with the product of phosphorylation of a sample of the
resolved diol of known absolute configuration: Vacca, J. P.; deSolms, S. J.;
Huff, J. R.; Billington, D. C.; Baker, R.; Kulagowski, J. J.; Maver, I. M.
Tetrahedron 1989, 45, 5679; 1991, 47, 907 (corrigenda).
(9) Baker, G. R.; Billington, D. C.; Gani, D. Bioorg. Med. Chem. Lett.
1991, 1, 17.
(10) Dielh, R. E.; Whiting, P.; Potter, J.; Gee, N.; Ragan, C. I.; Linemeyer,
D.; Schoepfer, R.; Bennet, C.; Dixon, R. A. F. J. Biol. Chem. 1990, 265,
5946.
(11) (a) Arnold, J. P. R.; Lowe, G. J. Chem. Soc., Chem. Commun. 1986,
865. (b) Arnold, J. P. R.; Bethell, R. C.; Lowe, G. Bioorg. Chem. 1987, 15,
250.
(12) The predicted intensity ratios for the isotopomers arising from the
configurational analysis of PSi from the enzyme-catalyzed reaction assuming
either inversion or retention can be calculated from the measured isotopic
incorporation. Mass spectrometry of D-1-Sp-myo-inositol [17O]thiophosphate
established that the actual 17O incorporation was 30%, with the remaining
isotope composition being 69% 16O and 1% 18O. For the 18O incorporation
during the enzymatic hydrolysis, this was estimated to be 83%, with the
remaining isotope composition 1% 17O and 16% 16O. Using these values, the
predicted ratio for the resonances from the trans ester (A:B:C) for inversion
of configuration is 40.6:34.9:24.5, whereas for retention of configuration the
ratios would be 40.6:24.5:34.9. The experimental ratios A:B:C of 40.5:33.5:
26 are in excellent agreement with complete inversion of configuration (the
ratios for the cis esters are, of course, complementary).
Extensive stereochemical studies on a wide range of phospho-
transferases have established that single-step reactions occur with
inVersion of configuration, consistent with an in-line displacement
† The University of Leicester.
‡ Manchester University.
§ University of Southampton.
(1) Majerus, P. W. Annu. ReV. Biochem. 1992, 61, 225.
(2) Potter, B. V. L.; Lampe, D. Angew. Chem., Int. Ed. Engl. 1995, 34,
1933.
(3) Atack, J. R. Brain Res. ReV. 1996, 22, 183.
(4) (a) Bone, R.; Springer, J. P.; Atack, J. R. Proc. Natl. Acad. Sci. U.S.A.
1992, 89, 10031. (b) Pollack, S. J.; Atack, J. R.; Knowles, M. R.; McAllister,
G.; Ragan, C. I.; Baker, R.; Fletcher, S. R.; Iversen, L. L.; Broughton, H. B.
Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5766. (c) Bone, R.; Frank, L.; Springer,
J. P.; Pollack, S. J.; Osborne, S.; Atack, J. R.; Knowles, M. R.; McAllister,
G.; Ragan, C. I.; Broughton, H. B.; Baker, R.; Fletcher, S. R. Biochemistry
1994, 33, 9460. (d) Bone, R.; Frank, L.; Springer, J. P.; Atack, J. R.
Biochemistry 1994, 33, 9468.
(5) Takimoto, K.; Okada, M.; Matsuda, Y.; Nakagawa, H. J. Biochem.
(Tokyo) 1985, 98, 363.
(6) (a) Leech, A. P.; Baker, G. R.; Shute, J. K.; Cohen, M. A.; Gani, D.
Eur. J. Biochem. 1993, 212, 693. (b) Baker, G. R.; Gani, D. Bioorg. Med.
Chem. Lett. 1991, 1, 193. (c) Cole, A. G.; Gani, D. J. Chem. Soc., Perkin
Trans. 1 1995, 2685. (d) Cole, A. G.; Wilkie, J.; Gani, D. J. Chem. Soc.,
Perkin Trans. 1 1995, 2695. (e) Wilkie, J.; Cole, A. G.; Gani, D. J. Chem.
Soc., Perkin Trans. 1 1995, 2706.
10.1021/ja991939r CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/31/1999

8386 J. Am. Chem. Soc., Vol. 121, No. 36, 1999
Communications to the Editor
Scheme 1a
Scheme 2. Outline of the Configurational Analysis of
Inorganic [16O,17O,18O]Thiophosphate, Shown for the Rp
Enantiomer That Would Arise if the Enzymatic Step
Proceeded with Inversion of Configurationa
a Only the isotopomers in which 17O is lost during the cyclization step
are shown since these are the only species with sharp resonances in the
high-field 31P NMR spectrum. Reagents: (i) recombinant bovine myo-
inositol monophosphatase, H218O (97% enriched), pH 9.0, 25 °C; (ii) (S)-
2-iodo-1-phenylethanol, DMF; (iii) (PhO)2P(O)Cl (1.1 equiv), tri-n-
butylamine, DMF; (iv) CH2N2, CH3CN.
a Reagents: (i) NaH, DMF, (2R,4R,5S)-(+)-2-chloro-3,4-dimethyl-5-
phenyl-1,3,2-oxazaphospholidine-2-sulfide (Aldrich); (ii) HPLC separation
of diastereomers; (iii) THF, (CF3CO)2O, H217O (53% enriched); (iv) Na,
liquid NH3.
nucleophilic displacement reaction and rules out an adjacent
displacement reaction accompanied by pseudorotation. These
observations are consistent with one of the models for the
hydrolysis mechanism advanced by the Merck group on the basis
of their X-ray structural work.4 In this mechanism, the nucleophilic
water is coordinated to Mg2+-1 and is activated by Glu-70 and
Thr-95, allowing approach in-line with the inositol leaving group.
It is interesting to note that fructose 1,6-bisphosphatase also
utilizes two magnesium cations and is sensitive to lithium, and
this enzyme proceeds with inversion.13
This result is highly significant since it reestablishes and
reinforces the generalization that single-step phosphoryl-transfer
reactions proceed via in-line mechanisms. In addition, it has firmly
ruled out a mechanism proceeding by a phosphoenzyme inter-
mediate. To date, there are no examples of enzyme-catalyzed
displacement reactions occurring via an adjacent attack involving
a pseudorotation step. Indeed, in simple phosphate esters,
particularly anions (mono- and diesters), there are few examples
of simple chemical reactions involving pseudorotation.14 This
mechanism is principally encountered in displacement reactions
involving phosphate esters held in small rings, where the ring
dramatically stabilizes the trigonal bipyramidal intermediate.
Hence, it would appear that in-line nucleophilic displacement is
certainly the preferred pathway for phosphate mono- and diester
anions in both chemical and enzyme-catalyzed reactions. This
result may also assist in the design of inhibitors of inositol
monophosphatase.
Figure 1. 31P NMR spectrum (Bruker ARX400; 162 MHz) of the sample
from the configurational analysis of inorganic [16O,17O,18O]thiophosphate
derived from the hydrolysis of D-Sp-1-myo-inositol [17O]thiophosphate
in H218O, catalyzed by recombinant bovine myo-inositol monophosphatase.
Assignments of resonances A-F are given in Scheme 2. The spectrum
was recorded at 0.038 Hz per point digital resolution and processed
without resolution enhancement, with natural line widths of 0.27 Hz.
shown in Scheme 2. The high-field 31P NMR spectrum of the
resulting isotopomeric cyclic phosphothiolate triesters derived
from the configurational analysis sequence is shown in Figure
1.12 This establishes that the absolute configuration of the
enzymatically derived inorganic [16O,17O,18O]thiophosphate is Rp
and that the reaction has proceeded with inVersion of configu-
ration. This result is entirely consistent with the expected in-line
Acknowledgment. We are indebted to Mrs. Nicola Muir for supplying
protein and Dr. Gerald Griffiths for high-field NMR spectroscopy.
(13) Domanico, P. L.; Rahil, J. F.; Benkovic, S. J. Biochemistry 1985, 24,
1623.
(14) (a) Thatcher, G. R. J.; Kluger, R. H. AdV. Phys. Org. Chem. 1989,
25, 99. (b) Hall, C. R.; Inch, T. D. Tetrahedron 1980, 36, 2059.
JA991939R