3006 J. Am. Chem. Soc., Vol. 120, No. 13, 1998
Pratt and Hammar
E-I in this case, but it is certainly also possible that the pendent
erstwhile leaving group may prevent incursion of methanol into
the active site.12 More experiments are required to settle this
issue.
Scheme 3
Thus, the cyclic phosphate 1 represents the first member of
a new class of molecules that can interact with â-lactam-
recognizing molecules to generate inert complexes. As in the
interaction of â-lactams with DD-peptidases, the latter property
may result from its cyclic structure and tethered leaving group.
Synthetic elaboration of 1 to give enzyme specificity is clearly
conceivable.
1
respectively. Absorption versus time traces were fitted to Scheme 3
by means of the FITSIM program.14 The Km of cephalothin as a
substrate of the P99 â-lactamase was taken to be 20 µM.15 Over the
concentration range of 1 studied, no enzyme saturation was required
to fit the data.
The reaction of 1 with the P99 â-lactamase could also be monitored
by the decrease in fluorescence emission as 1 (λex ) 310 nm, λf ) 370
nm) was converted to 3. The reaction was studied under second-order
conditions (enzyme and 1 concentrations were 4.0 and 2.0 µM,
respectively), and the data were fitted to eq 1 by means of a nonlinear
least squares program.16
Experimental Section
Materials. The â-lactamase of Enterobacter cloacae P99 and the
TEM-2 â-lactamase from Escherichia coli W3310 were purchased from
the Centre for Applied Microbiology and Research (Porton Down,
Wiltshire, U.K.) and used as received. The Streptomyces R61 DD-
peptidase was the generous gift of Dr. J.-M. Fre`re of the University of
Liege, Belgium.
1-Chloro-4,5-benzo-2,6-dioxaphosphorinanone (3)-1-Oxide, (2).
This compound was prepared from salicylic acid and phosphorus
oxychloride essentially as described by Montgomery et al.8 It was
purified by short-path vacuum distillation followed by recrystallization
from carbon tetrachloride. After purification, the air (water)-sensitive
material had the following properties: mp 91-95 °C (lit.8 mp 90-93
°C); FT-IR (KBr, cm-1) 1792, 1611, 1286 (lit.7 1790, 1605, 1280 cm-1);
1H NMR (MeCN-d3, 300 MHz) δ (ppm) 7.44 (1H, d, J ) 8.0 Hz),
7.56 (1H, t, J ) 8.0 Hz), 7.94 (1H, t, J ) 8.0 Hz), 8.16 (1H, d, J ) 8.0
Hz). It was stored in a sealed container at -20 °C.
1-Hydroxy-4,5-benzo-2,6-dioxaphosphorinanone (3)-1-Oxide, (1).
The cyclic phosphoryl chloride 2 (500 mg, 2.3 mmol) was suspended
in 60 mL of anhydrous acetonitrile (Aldrich, Sure/Seal) and water (4l
µL, 2.3 mmol) added with magnetic stirring. The mixture was then
gently warmed until the crystals of the starting material disappeared
(ca. 5 min). Two molar equivalents of redistilled dicyclohexylamine
(913 µL) were then added. The immediately precipitating solid
(dicyclohexylamine hydrochloride) was removed by vacuum filtration,
and the remaining solvent was removed by rotary evaporation. The
residual colorless solid was recrystallized from acetonitrile/diethyl ether
yielding 310 mg (35%) of the required product. Characterization: mp
160-163 °C; 1H NMR (2H2O, 300 MHz) δ (aromatics, ppm) 7.27 (d,
1H, J ) 8.0 Hz), 7.35 (t, 1H, J ) 8.0 Hz), 7.78 (t, 1H, J ) 8.0 Hz),
8.05 (d, 1H, J ) 8.0 Hz); 31P (2H2O) δ (ppm) -12.55; UV (20 mM
MOPS buffer, pH 7.5), λmax 239 nm (ꢀ ) 1.01 × 104 cm-1 M-1), 295
nm (ꢀ ) 2.52 × 103 cm-1 M-1); IR (KBr, cm-1) 1737, 1613, 1286;
ESMS (H2O, m/z) 563.1 (M + 2 C12H13N + H+). Anal. Calcd for
C19H28NO5P: C, 59.80; H, 7.40; N, 3.67; P, 8.12. Found: C, 60.11;
H, 7.44; N, 3.70; P, 7.91.
(1 - Io/Eo) exp[-ki(Eo - Io)t]
F ) F∞ + (Fo - F∞)
(1)
1 - (Io/Eo) exp[-ki(Eo - Io)t]
In eq 1, F is the fluorescence intensity at any time, Fo and F∞ are
the zero and infinite time intensities, respectively, and Io and Eo are
the initial concentrations of 1 and enzyme, respectively.
The rate of breakdown of the complex of 1 and the P99 â-lactamase
to free enzyme (kr, Scheme 2) was obtained by dilution of reaction
mixtures containing enzyme (0.8 µM) and 1 (100 µM) into a saturating
(0.94 mM) substrate (benzylpenicillin) solutions. The rate of turnover
of benzylpenicillin (monitored spectrophotometrically at 240 nm)
increased with time from close to zero to that characteristic of free
enzyme. The absorption versus time traces were fitted to eq 2
A ) Ao + V∞t - [(V∞ - Vo)/kr](1 - e-k t
)
(2)
r
where A and Ao are the absorbance at any time and at zero time,
respectively, and Vo and V∞ are the observed initial and final rates of
absorption change. The data were fitted to this equation by the
nonlinear least-squares procedure.16 Similar values of kr were obtained
as from the data fitted to Scheme 3.
Inactivation of the TEM enzyme was monitored by assaying the
activity of a reaction mixture (enzyme and 1 concentrations 0.4 and
100 µM, respectively) against benzylpenicillin (0.94 mM) as a function
of time. Initial rates decreased in a first-order fashion. The second-
order rate constant was obtained by dividing the pseudo-first-order
constant by the concentration of 1.
The rate constant of reactivation of the TEM â-lactamase was
determined in the same way as that of the P99 enzyme. The initial
enzyme and 1 concentrations were 0.4 and 100 µM, respectively.
The rate constants for inactivation and reactivation of the R61 DD-
peptidase were obtained by the general method employed for the TEM
â-lactamase. Enzyme (2 µM) and 1 (2 mM) were incubated together
and the concentration of the former with time determined by assay
against the depsipeptide substrate m-carboxyphenyl phenaceturate10 (3
mM). Reactivation was studied by means of the continuous monitoring
of the rate of hydrolysis of the same substrate after dilution out of 1.
Analytical and Kinetic Methods. The concentrations of stock
enzyme solutions were determined spectrophotometrically.13 All
enzyme kinetics experiments were performed at 25 °C in 20 mM MOPS
buffer, pH 7.5.
Second-order rate constants of inhibition of the P99 â-lactamase were
determined from absorption traces (270 nm) showing turnover of the
substrate cephalothin in the presence of enzyme and 1. Initial rates
decreased with time to a slow steady-state level when most of the free
enzyme had been converted into the inert complex. Initial concentra-
tions of enzyme, cephalothin and 1 were 1, 100, and 5-15 µM,
Acknowledgment. This research was supported by National
Institutes of Health grant AI 17986.
JA973313B
(12) This phenomenon occurs for example with the class A PCl
â-lactamase where the acyl enzymes derived from acyclic substrates are
susceptible to methanolysis while those from penicillins are not.10
(13) Xu, Y.; Soto, G.; Hirsch, K. R.; Pratt, R. F. Biochemistry 1996, 35,
3595-3603.
(14) Zimmerle, C. T.; Frieden, C. Biochem. J. 1989, 258, 381-387.
(15) Pazhanisamy, S.; Pratt, R. F. Biochemistry 1989, 28, 6870-6875.
(16) Johnson, M. L.; Halvorson, H. R.; Ackers, G. K. Biochemistry 1976,
15, 5363-5371.