C O M M U N I C A T I O N S
Scheme 2
S1, Supporting Information). Lys73 remains hydrogen bonded to
Tyr150 Oú. Coordinates of the structure have been deposited in
the RCSB protein data bank as entry 2P9V.
The mechanism of inhibition of the P99 â-lactamase by 4 and 5
can thus, from the data available at present, be represented by the
sequence shown in Scheme 2. This represents a novel cross-linking
of the active site and a previously unobserved specific modification
of one of the two conserved lysine residues of the â-lactamase active
site. Inhibition of class A â-lactamases by clavulanic acid and
penicillin sulfones has been shown to involve cross-linking of the
active site serine to the conserved Ser130.11,12
We have also observed that 4 and 5, and other derivatives of
these compounds, inhibit the class A TEM â-lactamase. We plan
further experiments to determine the scope of these compounds
against â-lactam-recognizing enzymes.
Figure 3. Top: crystal structure of the active site of the AmpC â-lactamase
after inhibition by 4, showing the carbamate cross-link between Ser64 and
Lys315. The electron density is contoured at the 3σ level. Bottom: the
same view of the wild-type enzyme,10 showing a clear gap between Ser64
and Lys315.
Acknowledgment. This research was supported by the National
Institutes of Health through Grant AI 17986 to R.F.P. and GM
63815 to B.K.S. K.B. is supported by a Ruth L. Kirschstein National
Research Service Award fellowship (GM 076883).
stituent, is not a better inhibitor than 4. This result is contrary to
what would be expected from comparable substitution in the
depsipeptides 1. A m-carboxy group in 1 is thought to interact
specifically with the P99 active site.8 The results suggest that 1
and 5 may not bind to the active site in the same way. It should be
noted, however, that inactivation of the enzyme by 5 was
competitively inhibited by p-nitrobenzene boronic acid, which is
itself a competitive inhibitor of the P99 enzyme.9
Supporting Information Available: Synthetic procedures for
compounds 4 and 5 and the kinetics methods. Details of the crystal-
lographic procedures and statistics are also provided. This material is
An electrospray mass spectrum of the inhibited enzyme was
obtained. Enzyme (10 µM) and 4 (5 mM) were incubated together
in MOPS buffer (above) for 5 min, after which time the enzyme
was inactive. The protein was then precipitated with trichloroacetic
acid, washed, and dried, and an ES+ mass spectrum obtained. The
spectrum showed an increase in protein mass of 29, in good
agreement with the mechanism of inactivation described below.
A 1.8 Å resolution crystal structure of the inhibited AmpC class
C â-lactamase was also obtained, as described in the Supporting
Information. The only observable difference from the structure of
the native enzyme10 was at the active site. In monomer A of the
structure, the Oγ oxygen of Ser64 is flipped some 180°
(Ser64CRCâOγC) and forms part of an unprecedented carbamate
bridge to Nê of Lys315 (Figure 3). Tyr150 has moved aside slightly
to accommodate insertion of a carbonyl, but Oú remains within
hydrogen-bonding distance of the inserted carbonyl oxygen (Figure
References
(1) Georgopapadakou, N. Exp. Opin. InVest. Drugs 2004, 13, 1307.
(2) Buynak, J. D. Biochem. Pharmacol. 2006, 31, 930.
(3) Pratt, R. F.; Govardhan, C. P. Proc. Natl. Acad. Sci. U.S.A. 1984, 81,
1302.
(4) Govardhan, C. P.; Pratt, R. F. Biochemistry 1987, 26, 3385.
(5) Cabaret, D.; Garcia Gonzalez, M.; Wakselman, M.; Adediran, S. A.; Pratt,
R. F. Eur. J Org. Chem. 2001, 141.
(6) Bell, J. H.; Pratt, R. F. Biochemistry 2002, 41, 4329.
(7) Renfrow, W. B., Jr.; Hauser, C. R. J. Am. Chem. Soc. 1937, 59, 2308.
(8) Ahn, Y.-M.; Pratt, R. F. Bioorg. Med. Chem. 2004, 12, 1539.
(9) Nagarajan, R.; Pratt, R. F. Biochemistry 2004, 43, 9664.
(10) Usher, K. C.; Blaszczak, L. C.; Weston, G. S.; Shoichet, B. K.; Remington,
S. J. Biochemistry 1998, 37, 16082.
(11) Brown, R. P. A.; Aplin, R. T.; Schofield, C. J. Biochemistry 1996, 35,
12421.
(12) Kuzin, A. P.; Nukaga, M.; Nukaga, Y.; Hujer, A.; Bonomo, R. A.; Knox,
J. R. Biochemistry 2001, 40, 1861.
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