The perfect penicillin? Inhibition of a bacterial DD-peptidase by peptidoglycan-mimetic β-lactams

Article abstract of DOI:10.1021/ja048850s

6-(Glycyl-L-α-aminopimelyl)-aminopenicillanic acid and 7-(glycyl-L-α-aminopimelyl)-aminocephalosporanic acid have been synthesized as Streptomyces sp. peptidoglycan-mimetic β-lactams. These compounds inactivate the Streptomyces R61 DD-peptidase with rate

Full text of DOI:10.1021/ja048850s

Published on Web 06/09/2004
The Perfect Penicillin? Inhibition of a Bacterial DD-Peptidase by
Peptidoglycan-Mimetic â-Lactams
Helen R. Josephine, Ish Kumar, and R. F. Pratt*
Department of Chemistry, Wesleyan UniVersity, Lawn AVenue, Middletown, Connecticut 06459
Received March 1, 2004; E-mail: rpratt@wesleyan.edu
The inactivation of bacterial DD-peptidases by â-lactams is the
reaction responsible for the antibiotic action of the latter mol-
ecules.1,2 Although a large number of â-lactams have been tested
for antibiotic activity and, in general, the most effective of them
have been chosen for clinical application, the relationship between
the â-lactam structure and antibiotic activity is not generally
predictable because of the large number of contributing factors.
Even the structural basis for the specificity of the â-lactam for the
target DD-peptidase is not well understood. One might imagine,
following the proposal of Tipper and Strominger,2 that good peptide
substrates of DD-peptidases and good â-lactam antibiotics would
have side-chains resembling those of the stem peptides of bacterial
peptidoglycan biosynthesis. There is, however, to date, little direct
evidence in favor of this idea and, curiously, a number of indications
to the contrary.3,4
Figure 1. Titration of 2 against the R61 DD-peptidase. The graph shows
residual activity against 1 after incubation of the enzyme (0.4 µM) with 2
(0-0.5 µM).
Recently, research in this laboratory has demonstrated that
peptide 1 is an excellent substrate of the Streptomyces R61 DD-
peptidase and the best peptide substrate yet discovered for a DD-
peptidase, with a kcat/Km value of 8.7 × 106 s-1 M-1 5
.
the DD-peptidase is inactivated by 2 in a stoichiometric 1:1 fashion,
as anticipated for the reaction of a â-lactam with a DD-peptidase.
This peptide is distinguished by the presence of an amine terminus
characteristic of the stem peptide of Streptomyces sp. Although
studies with less specific substrates of this enzyme have suggested
that side-chain specificity may be different in peptide substrates
than in â-lactam inhibitors,3,6-9 the question of the relative reactivity
of peptides and â-lactams that contain highly specific side-chains
has not yet been directly addressed. In this paper, we describe the
synthesis of the penicillin 2 and the cephalosporin 3 (Supporting
Information), each bearing the Streptomyces-specific side chain
present in 1.
Measurement of the second-order rate constant for the inactiva-
tion, ki, could not be made directly by manual mixing methods or
by competition with classical â-lactams such as cephalothin because
the reaction was too rapid. Finally, measurement of the rate constant
with good precision, was found to be possible from competition
experiments employing the specific chromophoric thiolester 4 as
the substrate (kcat ) 42 s-1, Km ) 4.0 µM, kcat/Km ) 1.05 × 107
s-1 M-1).
Figure 2A shows spectrophotometric total progress curves for
reaction of the DD-peptidase with 4 in the absence and presence
of 2; in the latter case, 2 inactivates the enzyme prior to complete
turnover of 4. These curves, taken from a series of experiments at
several concentrations of 2 and 4, could then be fitted to Scheme
1 by the Dynafit program10 to yield a ki value of (1.0 ( 0.2) × 107
s-1 M-1. The rate constant for inactivation could also be obtained
fluorimetrically11 in a stopped flow experiment. Figure 2B shows
the result of such an experiment. A series of such runs gave a value
of ki of (2.0 ( 0.4) × 107 s-1 M-1, in good agreement with the
value obtained from the competition experiment described above.
The data presented below show that 2 is an excellent inhibitor
of the R61 DD-peptidase, with a second-order inactivation rate
constant of 1.5 × 107 s-1 M-1. This represents, to our knowledge,
the largest rate constant yet reported for inactivation of a DD-
peptidase by a â-lactam and directly addresses the question referred
to above.
Compounds 2 and 3 were found to rapidly inactivate the
Streptomyces R61 DD-peptidase. From assays of mixtures of the
enzyme with increasing concentrations of 2, for example, the
titration curve shown in Figure 1 was obtained. This indicates that
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C O M M U N I C A T I O N S
selected for interbacterial warfare. Perhaps they proved to be too
dangerous to handle? Or is there an elegant defense?
The results described above show that 2 and 3 are extremely
potent inactivators of the Streptomyces R61 DD-peptidase. For
perspective, benzylpenicillin, the hitherto reported most effective
â-lactam inhibitor of this enzyme, reacts with a rate constant of
1.37 × 104 s-1 M-1 and the cephalosporin cephalothin with a rate
constant of 1.4 × 103 s-1 M-1.6 Thus, it is clear that, at least in the
case of this DD-peptidase, a peptidoglycan-mimetic side chain
dramatically improves the effectiveness of a â-lactam inhibitor as
it does a peptide substrate.5 It is possible that the reactions of both
1 and 2 with the enzyme are diffusion-limited. It was not possible
to readily test this proposition by a direct viscosity experiment since
it appeared that both glycerol and sucrose significantly depressed
the reactivity of the enzyme against both benzylpenicillin and
cephalothin; these latter reactions cannot be diffusion-controlled.
It should be noted that â-lactams with peptide and peptidoglycan-
mimetic side-chains have previously been reported in the litera-
ture.15,16 In general, they have been found to not be good substrates.
There are, as noted above, many reasons why this might be so, but
in no case to date has their reactivity with a specific target enzyme
been studied. On the other hand, there is evidence that nonspecific
peptide side-chains on â-lactams yield poor DD-peptidase inhibi-
tors.17 At any event, there is now one unambiguous case where
incorporation of a specific peptidoglycan-mimetic side chain into
a â-lactam has had a dramatic positive effect on its reactivity with
a DD-peptidase. It will be of interest to explore the generality of
this result and to consider its implications to the further design of
DD-peptidase inhibitors.
Figure 2. (A) Total progress curve for hydrolysis of the substrate 4 (300
µM) by the R61 DD-peptidase (30 nM) in the absence (filled circles) and
presence (filled squares) of 2 (40 nM). The reaction was monitored
spectrophotometrically at 240 nm. (B) Total progress curve for direct
reaction of 2 (6 µM) with the R61 DD-peptidase (1.67 µM). The reaction
was monitored fluorimetrically at 324 nm.
Acknowledgment. This research was supported by the National
Institutes of Health through Grant AI 17986 to R.F.P. We are
grateful to Dr. J.-M. Fre`re of the University of Lie`ge, Lie`ge,
Belgium, for supplies of the R61 DD-peptidase.
No indication was observed of enzyme saturation by the inhibitor
at the concentrations employed (e6.0 µM).
Supporting Information Available: Synthetic procedures for
compounds 2 and 3, as well as kinetics methods (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
Scheme 1
References
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1133.
The rate constant for inactivation of the enzyme by the
cephalosporin 3 was also determined from competition experiments
with the substrate 4; the value of ki thus obtained was (5.6 ( 1.5)
× 105 s-1 M-1. The R61 DD-peptidase is usually more susceptible
to penicillins than to cephalosporins.6
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a good substrate of the class C Enterobacter cloacae P99 â-lac-
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suggested,13 and as Meroueh et al.14 also noted recently, â-lac-
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â-lactams with peptidoglycan-mimetic side-chains have not been
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