Inhibition of Serine â-Lactamases by Acyl Phosph(on)ates
J. Am. Chem. Soc., Vol. 120, No. 18, 1998 4265
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Experimental Section
Enzymes and Substrates. The â-lactamases of the TEM-2 plasmid
and from E. cloacae P99 were purchased from the Centre for Applied
Microbiology and Research (Porton Down, Wilts, U.K.) and used as
supplied. Cephalothin was a gift from Eli Lilly and Co. Benzylpeni-
cillin was purchased from Sigma Chemical Co.
Essentially the same procedure was used to obtain rates of hydrolysis
of transient intermediates in the cases of 3-5 with the P99 enzyme
and of 5 with TEM. The velocity of cephalothin hydrolysis increased
with time due to hydrolysis of the intermediate. Rate constants for
this hydrolysis were obtained by the fitting of absorbance data to eq 2,
Synthesis of Acyl Phosph(on)ates. Sodium Benzoyl Phenyl
Phosphate 3. This compound was prepared by a modification of the
method employed by Jencks and Carriuolo for the synthesis of acetyl
phenyl phosphate.17 Thus, to a solution of 2.0 g (7.8 mmol) of disodium
phenyl phosphate (Aldrich Chemical Co.) in 15 mL of water, cooled
in an ice bath, was added 3.5 g (15.8 mmol) of benzoic anhydride
(Acros Organics) dissolved in 10 mL of pyridine, dropwise with stirring.
After 25 min, the reaction mixture was extracted three times with diethyl
ether and the aqueous phase then freeze-dried. The solid residue was
recrystallized twice from water and characterized by NMR spectra: 1H
(2H2O) δ 7.27 (t, J ) 7.5 Hz, 1H), 7.29 (d, J ) 7.5 Hz, 2H), 7.44 (t,
J ) 7.5 Hz, 2H), 7.60 (t, J ) 7.5 Hz, 2H), 7.75 (t, J ) 7.5 Hz, 1H),
8.10 (d, J ) 7.5 Hz, 2H); 31P (2H2O) δ -14.4. No resonances other
than these were observed in the spectra. The compound was thus at
least 95% pure, with respect to its organic and phosphorus content.
The presence of impurities below this level however cannot give rise
to the observations described below.
Sodium Benzoyl Phenylphosphonate 4. The procedure of Laird
and Spence18 appeared, in our hands, to yield the diester dibenzoyl
phenylphosphonate, mp 104-106 °C (recrystallized from acetoni-
trile): 1H NMR (C2HCl3) δ 7.50 (t, J ) 7.5 Hz, 4H), 7.59 (t, J ) 7.5
Hz, 2H), 7.68 (m, 3H), 8.13 (d, J ) 7.5 Hz, 4H), 8.15 (m, 2H); 31P
NMR (C2HCl3) δ 7.20. This diester was dissolved in 1:1 acetone/
water and titrated to a stable pH 7 endpoint over 30 min with sodium
bicarbonate. Acetone was then removed by rotary evaporation and the
residual aqueous solution freeze-dried. The required product was
purified by elution with water from a Biorad P-2 column: 1H NMR
(2H2O) δ 7.54 (t, J ) 7.5 Hz, 2H), 7.55-7.65 (m, 3H), 7.67 (t, J ) 7.5
Hz, 1H), 7.87 (dd, J ) 7.5, 13.8 Hz, 2H), 8.10 (d, J ) 7.5 Hz, 2H);
31P NMR (2H2O) δ 9.80. This compound was also pure to the same
degree as 3.
Sodium Dibenzoyl Phosphate 5. This compound was prepared
analogously to 3, beginning with disodium hydrogen phosphate and
benzoic anhydride in a 1:2 molar ratio. The product was recrystallized
twice from water, with a final melting point of 195-197 °C: 1H NMR
(2H2O) 7.55 (t, J ) 7.5 Hz, 4H), 7.72 (t, J ) 7.5 Hz, 2H), 8.09 (d, J
) 7.5 Hz, 4H); 31P NMR (2H2O) δ -17.9. Anal. Calcd for C14H10-
NaO6P: C, 51.24; H, 3.07; P, 9.44. Found: C, 51.46; H, 2.80; P, 9.29.
Analytical and Kinetic Methods. The concentrations of stock
enzyme solutions were determined spectrophotometrically.19 Steady
state kinetic parameters were directly obtained for 3 and 5 with the
P99 â-lactamase and for 3 and 4 with TEM by the method of
Wilkinson20 from spectrophotometric initial velocity measurements. A
Hewlett-Packard HP8452A spectrophotometer was routinely employed.
The wavelengths employed were 244 nm (∆ꢀ ) 8720 cm-1 M-1), 252
nm (∆ꢀ ) 2375 cm-1 M-1), and 248 nm (∆ꢀ ) 14 440 cm-1 M-1) for
3-5, respectively. All kinetics experiments were performed at 25 °C
in 20 mM MOPS buffer, pH 7.5.
Second-order rate constants of irreversible inhibition were obtained
from incubation mixtures of appropriate concentrations of enzyme and
inhibitor where that of the latter much exceeded that of the former
(pseudo-first-order conditions). Aliquots of these were diluted into
assay mixtures containing the substrate cephalothin at saturating
concentration (1 mM), and the residual enzyme activity was determined
spectrophotometrically from the initial rates of substrate turnover. Rate
constants were then obtained from eq 1 where V is the initial rate of
cephalothin consumption in the assay at any time, V0 the rate at time
zero, ki the second-order rate constant of inactivation, and Io the inhibitor
concentration.
returnt
A ) Ao - V∞t + (Vo/kreturn)(1 - e-k
)
(2)
where Ao is the initial absorbance, Vo the initial rate, V∞ the final rate
after return of activity was complete, and kreturn the rate constant for
return of activity. In the cases of 4 with the P99 enzyme and 5 with
TEM, a more complicated reaction scheme involving competing
turnover and inhibition was required (see Scheme 1). To obtain rate
constants (including kcat and Km) under these circumstances, the
absorbance of the inhibitor with time in the presence of the enzyme
was monitored and the data were analyzed by means of the FITSIM
program.21
Results and Discussion
Acyl phosph(on)ates such as 3-5 can be readily prepared,
e.g., see the Experimental Section, and form convenient stable
salts. They are remarkably stable at neutral pH22-24 (pseudo-
first-order rate constants of hydrolysis of 3-5 in 20 mM MOPS,
pH 7.5, were 1.6 × 10-7, 8.3 × 10-6, and 7.8 × 10-7 s-1
,
respectively; the comparable value for benzylpenicillin is 1.5
× 10-5 s-1) but more labile at alkaline pH, presumably through
nucleophilic attack at the carbonyl group;23 simple alkyl and
aryl phosph(on)ate monoanions are extremely stable to nucleo-
philic cleavage in alkaline solution. Second-order rate constants
of alkaline hydrolysis of 3-5 were 0.32, 0.090, and 1.5 s-1
M-1, respectively. These rate constants can be compared with
that for benzylpenicillin, 0.1 s-1 M-1. The acyl phosph(on)-
ates are however quite labile to aminolysis by primary amines,22
as are penicillins.25
Typical class A and C â-lactamases, the TEM-2 â-lactamase,
and the â-lactamase of E. cloacae P99, respectively, were found
to catalyze the hydrolysis of 3-5 (and of other analogous acyl
phosph(on)ates) to benzoate and phosph(on)ate. The products
could be readily identified in 1H NMR spectra of reaction
mixtures. It is clear however from the kcat and Km values
presented in Table 1 that these compounds are poor substrates
of these enzymes, especially with respect to kcat, which, for good
substrates, may exceed 1000 s-1 (see data for benzylpenicillin
in Table 1). Nonetheless, it is a little surprising that compounds
as nonspecific in structure as 3-5 are substrates at all.
Previously studied ester substrates included â-lactamase-specific
amido side chains in their structures.19,26
Important questions of course in each case are whether the
enzyme is catalyzing an acyl or phosphor(on)yl transfer reaction
and then whether a covalent acyl or phosphor(on)yl enzyme
intermediate, respectively, is involved. These questions will
be addressed below.
Of greater interest than the above-mentioned steady-state
parameters was the observation that in several instances there
was evidence that the intermediates involved only slowly
(seconds to minutes) led back to free enzyme: when small
aliquots of reaction mixtures were added to assay solutions
(21) Zimmerle, C. T.; Frieden, C. Biochem. J. 1989, 258, 381-387.
(22) Chantrenne, H. Biochim. Biophys. Acta 1948, 2, 286-293.
(23) DiSabato, G.; Jencks, W. P. J. Am. Chem. Soc. 1961, 83, 4400-
4405.
(24) Kluger, R.; Tsui, W.-C. J. Org. Chem. 1980, 45, 2723-2724.
(25) Page, M. I. AdV. Phys. Org. Chem. 1987, 23, 165-270.
(26) Govardhan, C. P.; Pratt, R. F. Biochemistry 1987, 26, 3385-3395.
(17) Jencks, W. P.; Carriuolo, J. J. Biol. Chem. 1959, 234, 1272-1279.
(18) Laird, R. M.; Spence, M. J. J. Chem. Soc., Perkin Trans. 2 1973,
1434-1436.
(19) Xu, Y.; Soto, G.; Hirsch, K. R.; Pratt, R. F. Biochemistry 1996, 35,
3595-3603.
(20) Wilkinson, G. N. Biochem. J. 1961, 80, 324-332.