Fig. 2 Stereo views of the active site of PPE (in green) showing the N-benzoyl b-sultam (1) (in beige) covalently linked via a sulfonate ester to Ser-195 and
the ‘native’ position of His-57 in thin lines.
However, we have shown, for example, that b-lactamase is
almost as efficient at catalysing P–N fission in phosphonami-
dates as it is at increasing the rate of C–N cleavage in b-
lactams.18 It appears also that similar catalytic machinery is
used by elastase for both hydrolysis of peptides and for its
inactivation by b-sultams.
This work was supported by EPSRC CASE awards with
British Biotech (P. S. H.) and with AstraZeneca (J. M. W).
Notes and references
1 P. R. Bernstein, P. E. Edwards and J. C. Williams, Prog. Med. Chem.,
1994, 31, 59.
2 W. Bode, A. Z. Wei, R. Huber, E. Meyer, J. Travis and S. Neuman,
Fig. 1 The pH dependence of the rate constants kcat/Km, for the hydrolysis
of N-succinyl- -(ala)3-p-nitroanilide by PPE (5 Left Hand scale), and ki, for
the inactivation of PPE by N-benzoyl b-sultam (: Right Hand scale).
EMBO J, 1986, 5, 2453; M. A. Navia, B. M. McKeever, J. P. Springer,
T. Y. Lin, H. R. Williams, E. M. Fluder, C. P. Dorn and K. Hoogsteen,
Proc. Natl. Acad. Sci. USA, 1989, 86, 7.
L
3 E. Meyer, G. Cole, R. Radhakrishnan and O. Epp, Acta Crystallogr.,
Sect. B, 1988, 44, 26.
4 L. H. Takahashi, R. Radhakrishan, R. E. Rosenfeld, Jr., E. F. Meyer, Jr.
and D. A. Trainer, J. Am. Chem. Soc., 1989, 111, 3368; A. Renaud, P.
Lestienne, D. L. Hughes, J. G. Bieth and J.-L. Dimicoli, J. Biol. Chem.,
1983, 258, 8312.
5 M. J. Costanzo, B. E. Maryanoff, L. R. Hecker, M. R. Schott, S. C.
Yabut, H.-C. Zhang, P. Andrade-Gordon, J. A. Kauffman, J. M. Lewis,
R. Krishnan and A. Tulinsky, J. Med. Chem., 1996, 39, 3039; P. D.
Edwards, D. W. Andisik, A. M. Strimpler, B. Gomes and P. A. Tuthill,
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6 S. J. F. McDonald, G. D. E. Clarke, M. D. Dowle, L. A. Harrison, S. T.
Hodgson, G. G. A. Inglis, M. R. Johnson, P. Shah, R. J. Upton and S. B.
Walls, J. Org. Chem., 1999, 64, 5166.
7 M. I. Page, ed. The Chemistry of b-Lactam Antibiotics, Blackie,
Glasgow, 1992.
8 R. Chabin, B. G. Green, P. Gale, A. L. Maycock, H. Weston, C. P. Dorn,
P. E. Finke, W. K. Hagmann, J. J. Hale, M. MacCross, S. K. Shah, D. J.
Underwood, J. B. Doherty and W. B. Knight, Biochemistry, 1993, 32,
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9 D. J. Underwood, B. G. Green, R. Chabin, S. Mills, J. B. Doherty, P. E.
Finke, M. MacCoss, S. K. Shah, C. S. Burgey, T. A. Dickinson, P. R.
Griffin, T. E. Lee, K. M. Swiderek, T. Covey, W. M. Westler and W. B.
Knight, Biochemistry, 1995, 34, 143 344.
10 R. C. Wilmouth, S. Kassamally, N. J. Westwood, R. J. Sheppard,
T. D. W. Claridge, R. T. Aplin, P. A. Wright, G. J. Pritchard and C. J.
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disorder. An alternative possibility is that the amide bond has
been hydrolysed either before or after ring-opening by Ser-195,
but there was no evidence for this in the ESI-MS analysis.
One oxygen atom of the sulfonate is located in the oxyanion
hole within hydrogen bonding distance of the amido-nitrogen
atoms of Ser-195 (3.02 Å) and Gly-193 (3.23 Å). The other
sulfonate oxygen atom is located in the upper part of the S1
pocket. It is worth noting that the native enzyme crystallises
with a sulfate ion in the oxyanion hole. The other atoms of the
ring-opened b-sultam are located in the P1/P2 region. The side
chain of His-57 has been displaced by approximately 90° from
its normal location and to a position similar to that observed in
the structure of a g-lactam inhibitor bound to PPE.15 The
electron density map is consistent with the presence of two
water molecules (Wat-342 and Wat-355) occupying the ‘native’
position of His-57.16 Another water molecule in the vicinity
(Wat-371) is located in a similar position to the hydrolytic water
observed in the structure of an acyl-enzyme intermediate
between PPE and a peptide inhibitor.17
The electron density map indicates that the side chain of His-
57 clearly has extra density extending from its Ne2 atom. This
may reflect the sulfonation of His-57 by another molecule of the
b-sultam inhibitor consistent with the ESI-MS results. However
the ‘extra’ electron density is not sufficiently well-defined for
any atoms to be built in. We are improving the selectivity of
inactivation by synthesising b-sultams with suitably placed
substituents.
11 M. I. Page, in Comprehensive Medicinal Chemistry, vol. 2, ed. P G
Sammes, Pergamon, Oxford, 1990, pp 61–87.
12 J. F. King, R. Rathore, J. Y. L. Lam, L. E. R. Gao and D. F. Klassen,
J. Am. Chem. Soc., 1992, 114, 3028.
It is generally accepted that nucleophilic substitution at acyl
centres proceeds through the formation of an unstable tetra-
hedral intermediate (TI). Furthermore, it is assumed that there is
some preferential direction of nucleophilic attack such that the
incoming nucleophile approaches at approximately the tetra-
hedral angle to the carbonyl group. By contrast, the mechanism
for sulfonyl group transfer often involves a pentacoordinate
intermediate or transition state with trigonal bipyramidal
geometry.13 It is often assumed, but with little actual supporting
evidence, that enzymes catalyse reactions by an exquisite
positioning of the catalytic groups. If this were the case then it
is doubtful if an enzyme with a primary function, say, as a
catalyst for acyl transfer could be an effective catalyst for
sulfonyl transfer because of these geometrical differences.
13 N. J. Baxter, A. P. Laws, L. J. M. Rigoreau and M. I. Page, J. Am. Chem.
Soc., 2000, 112, 3375.
14 J. Baxter, A. P. Laws, L. J. M. Rigoreau and M. I. Page, J. Chem. Soc.,
Perkin Trans. 2, 1996, 2245.
15 R. C. Wilmouth, S. Kassamally, N. J. Westwood, R. J. Sheppard,
T. D. W. Claridge, R. T. Aplin, P. A. Wright, G. J. Pritchard and C. J.
Schofield, Biochemistry, 1999, 38, 7989.
16 E. Meyer, G. Cole, R. Radhakrishnan and O. Epp, Acta Cryst., 1988,
B44, 26.
17 R. C. Wilmouth, I. J. Clifton, C. V. Robinson, P. L. Roach, R. T. Aplin,
N. J. Westwood, J. Hajdu and C. J. Schofield, Nat. Struct. Biol., 1997,
4, 456.
18 M. I. Page and A. P. Laws, Chem. Commun., 1998, 1609; M. I. Page,
Curr. Pharm. Des., 1999, 5, 895; M. J. Slater, A. P. Laws and M. I. Page,
Bioorg. Chem., in press.
498
Chem. Commun., 2001, 497–498