Nuances of mechanisms and their implications for evolution of the versatile β-lactamase activity: From biosynthetic enzymes to drug resistance factors

Article abstract of DOI:10.1021/ja963708f

β-Lactamases of classes A and C are believed to have evolved from bacterial enzymes involved in biosynthesis of the peptidoglycan, the so-called penicillin-binding proteins. All these enzymes undergo acylation at an active-site serine by β-lactam antibiotics as a common feature. However, the fate of the acyl-enzyme species is different for β-lactamases and penicillin-binding proteins; deacylation is rapid for the former, whereas it is slow for the latter. It is believed that the acquisition of the ability to deacylate the acyl-enzyme intermediate led to the evolution of β-lactamase activity, which is indispensable for the survival of bacteria in the face of challenge by β-lactam antibiotics. The mechanisms of deacylation of acyl-enzyme intermediates for β-lactamases are examined as a means to investigate structural factors in evolutionary descendency of classes A and C of β-lactamases from penicillin-binding proteins. It is known that in class A β-lactamases the hydrolytic water approaches the acyl-enzyme intermediate from the α-face, a process which is promoted by Glu-166 of these enzymes. An approach from the β-face for class C β-lactamase has been proposed. The process of activation of the hydrolytic water is not entirely understood at the present for these enzymes. Two compounds, p-nitrophenyl (2R,5R)-5-prolylacetate (2) and p-nitrophenyl (1S,3S)-3-carboxycyclopentylacetate (3), were synthesized as mechanistic probes to explore whether the hydrolytic water molecule actually approaches the acyl-enzyme species from the β-face and to investigate a notion that the ring amine at the acyl-enzyme intermediate may promote the hydrolytic reaction. Compound 2 acylates the active site serine of the Q908R β-lactamase (a class C enzyme), and the intermediate undergoes deacylation. On the other hand, compound 3 only acylates the active site, and not having the requisite amine in its structure, the intermediate resists deacylation. Both compounds serve as substrates for the class A TEM-1 β-lactamase, as they were expected, since the approach of the hydrolytic water molecule is from the a-face in this enzyme and is not promoted by the substrate itself. We conclude that substrate-assisted catalysis applies for the class C β-lactamases. On the basis of the evidence discussed, the knowledge of the crystal structures for the classes A and C of β-lactamases and the Streptomyces R61 DD-peptidase/transpeptidase (a PBP), it is proposed herein that evolution of classes A and C of β-lactamases from a primordial penicillin-binding protein should have been independent events; hence, the process does not represent a linear descendency of one β-lactamase from the other.

Full text of DOI:10.1021/ja963708f

VOLUME 119, NUMBER 33
AUGUST 20, 1997
© Copyright 1997 by the
American Chemical Society
Nuances of Mechanisms and Their Implications for Evolution
of the Versatile â-Lactamase Activity: From Biosynthetic
Enzymes to Drug Resistance Factors
Alexey Bulychev, Irina Massova, Kazuyuki Miyashita, and Shahriar Mobashery*
Contribution from the Department of Chemistry, Wayne State UniVersity,
Detroit, Michigan 48202
ReceiVed October 24, 1996X
Abstract: â-Lactamases of classes A and C are believed to have evolved from bacterial enzymes involved in
biosynthesis of the peptidoglycan, the so-called penicillin-binding proteins. All these enzymes undergo acylation at
an active-site serine by â-lactam antibiotics as a common feature. However, the fate of the acyl-enzyme species is
different for â-lactamases and penicillin-binding proteins; deacylation is rapid for the former, whereas it is slow for
the latter. It is believed that the acquisition of the ability to deacylate the acyl-enzyme intermediate led to the
evolution of â-lactamase activity, which is indispensable for the survival of bacteria in the face of challenge by
â-lactam antibiotics. The mechanisms of deacylation of acyl-enzyme intermediates for â-lactamases are examined
as a means to investigate structural factors in evolutionary descendency of classes A and C of â-lactamases from
penicillin-binding proteins. It is known that in class A â-lactamases the hydrolytic water approaches the acyl-
enzyme intermediate from the R-face, a process which is promoted by Glu-166 of these enzymes. An approach
from the â-face for class C â-lactamase has been proposed. The process of activation of the hydrolytic water is not
entirely understood at the present for these enzymes. Two compounds, p-nitrophenyl (2R,5R)-5-prolylacetate (2)
and p-nitrophenyl (1S,3S)-3-carboxycyclopentylacetate (3), were synthesized as mechanistic probes to explore whether
the hydrolytic water molecule actually approaches the acyl-enzyme species from the â-face and to investigate a
notion that the ring amine at the acyl-enzyme intermediate may promote the hydrolytic reaction. Compound 2
acylates the active site serine of the Q908R â-lactamase (a class C enzyme), and the intermediate undergoes deacylation.
On the other hand, compound 3 only acylates the active site, and not having the requisite amine in its structure, the
intermediate resists deacylation. Both compounds serve as substrates for the class A TEM-1 â-lactamase, as they
were expected, since the approach of the hydrolytic water molecule is from the R-face in this enzyme and is not
promoted by the substrate itself. We conclude that substrate-assisted catalysis applies for the class C â-lactamases.
On the basis of the evidence discussed, the knowledge of the crystal structures for the classes A and C of â-lactamases
and the Steptomyces R61 DD-peptidase/transpeptidase (a PBP), it is proposed herein that evolution of classes A and
C of â-lactamases from a primordial penicillin-binding protein should have been independent events; hence, the
process does not represent a linear descendency of one â-lactamase from the other.
The â-lactamase activity is the primary means for bacterial
resistance to â-lactam antibiotics. Of the four known classes
of â-lactamases, class A enzymes are most common among
pathogens, whereas class C enzymes are second most common.
On the basis of similarity of the three-dimensional fold for
â-lactamases of known structures with penicillin-binding pro-
teins (PBPs), enzymes involved in cellwall biosynthesis in
bacteria, these proteins are believed to be related by common
ancestry. Both classes A and C of â-lactamases, as well as the
known PBPs, undergo acylation at an active-site serine residue
* Author to whom correspondence should be addressed.
X Abstract published in AdVance ACS Abstracts, August 1, 1997.
S0002-7863(96)03708-0 CCC: $14.00 © 1997 American Chemical Society

7620 J. Am. Chem. Soc., Vol. 119, No. 33, 1997
BulycheV et al.
The product was purified by silica gel column chromatography
(methanol/chloroform/aqueous ammonia, 1/30/0.1) to afford the title
compound (76 mg; yield, 95%): mp 114 °C; Rf 0.28 (methanol/
chloroform/aqueous ammonia, 1/30/0.1); 1H NMR (CDCl3) δ 1.29 (9H,
s, CH3 t-Bu), 1.27-1.36 (1H, unresolved m, C3 or C4 methylene),
1.61-1.74 (1H, m, C3 or C4 methylene), 1.76-1.88 (1H, m, C3 or
C4 methylene), 1.98-2.11 (1H, m, C3 or C4 methylene), 2.34 (2H, d,
J ) 7 Hz, -CH2CO2R), 3.42-3.50 (1H, unresolved m, C5 methine),
3.51 (3H, s, CH3 methoxy), 3.61 (1H, dd, J ) 6 and 8 Hz, C2 methine),
6.54 (1H, broad s, amine); 13C NMR (CDCl3) δ 27.6, 29.1, 30.6, 40.1,
by â-lactam antibiotics. The rate of deacylation of this acyl-
enzyme intermediate from the active site of penicillin-binding
proteins is slow, thereby the bacterium is deprived of the
biosynthetic function of these enzymes, an event that results in
bacterial death. However, â-lactamases are capable of undergo-
ing deacylation in a facile manner, completing the turnover
necessary for hydrolysis of the â-lactam antibiotics.
Availability of crystal structures from the Brookhaven Protein
Data Bank for several of these proteins (two PBPs, three class
A â-lactamases, and one class C â-lactamase) has stimulated
research on the mechanisms of these important bacterial
enzymes. Despite the availability of the structural information,
the details of the catalytic mechanisms for all these proteins
remain elusive. Furthermore, evolutionary relationship among
these proteins is not known. Lobkovsky et al.1 have proposed
on the basis of analysis of crystal structures that class C
â-lactamases arose from PBPs and, subsequently, class A
enzymes arose from class C enzymes (linear evolution). Insofar
as all these proteins undergo acylation by â-lactam antibacterials,
we decided to investigate the processes for deacylation, both
to delineate the pertinent mechanisms, as well as to gain a
mechanistic handle on the subject of the evolutionary diversi-
fication of â-lactamases from PBPs. We disclose herein that
the classes A and C of â-lactamases have developed entirely
distinct approaches for catalyzing the deacylation step of their
respective acyl-enzyme intermediates. As it is inconceivable
to see how such disparate diversification in the catalytic
machinery can arise from a linear evolutionary process, we
conclude that this mechanistic information supports an alterna-
tive model for the evolution of this family of proteins. This
model states that the two classes of â-lactamases evolved
independently from the ancestral PBP(s).
51.1, 54.3, 59.5, 80.8, 172.0, 173.7; [R]25 +112° (c 0.805, ethyl
D
acetate); CI MS 244 (M + H, 45%).
(5R,2R)-1-N-(tert-Butoxycarbonyl)-5-(carboxymethyl)pyrrolidine-
2-carboxylic Acid tert-Butyl Ester (10). An aqueous solution of
NaOH (90 µL, 0.4 mmol) was added dropwise to the solution of 8
(100 mg, 0.4 mmol) in dioxane (7 mL) at ice-water temperature, and
the mixture was stirred at this temperature for 6 h. The solvent was
evaporated in Vacuo, giving a yellowish residue, which was dissolved
in water. The pH of the solution was adjusted to 2.0 by the addition
of concentrated HCl, and the product was extracted into ethyl acetate
(4×, 120 mL each). The combined organic fraction was dried over
anhydrous MgSO4, the mixture was filtered, and the filtrate was
evaporated to dryness to give a pale yellow solid (82 mg); mp 124 °C.
The product of the above reaction (152 mg, 0.65 mmol) was
dissolved in 10 mL of water/dioxane mixture (1/2), and the pH of the
solution was adjusted to 9.0 by the addition of 6 N NaOH. Di-tert-
butyl dicarbonate (214 mg, 0.98 mmol) was added to the mixture with
stirring at ice-water temperature, and the reaction was allowed to
progress at that temperature for 24 h. Dioxane was removed by
evaporation in Vacuo. The pH of the aqueous solution was adjusted
to 2.0 with concentrated HCl, and the product was extracted into ethyl
acetate (4×, 120 mL each). The combined organic fraction was dried
over anhydrous MgSO4, the solid was filtered, and the filtrate was
evaporated to dryness. The product was purified by silica gel column
chromatography (methanol/chloroform, 1/20) to afford the title com-
pound as a white solid (180 mg; yield, 83%): mp 104 °C; Rf 0.72
1
(methanol/chloroform, 1/20); H NMR (CDCl3) δ 1.40 (9H, s, t-Bu),
Experimental Section
1.43 (9H, s, t-Bu), 1.70-1.79 (1H, m, C3 or C4 methylene), 1.83-
1.94 (1H, m, C3 or C4 methylene), 2.02-2.11 (1H, m, C3 or C4
methylene), 2.15-2.25 (1H, m, C3 or C4 methylene), 2.41 (1H, dd, J
) 10 and 16 Hz, C6 methylene), 3.14 (1H, dd, J ) 4 and 16 Hz, -CH2-
CO2H), 4.09 (1H, m, C5 methine), 4.22 (1H, m, C2 methine), 10.41
(1H, broad s, carboxylic acid); 13C NMR (CDCl3) δ 27.9, 28.2, 28.7,
Hydrogen and carbon NMR spectra were obtained at 300 and 75
MHz, respectively, using a Gemini-300 Varian spectrometer, or at 500
and 125 MHz, respectively, using U-500 Varian spectrometer; chemical
shift values (δ) are given in parts per million. Infrared and mass spectra
were recorded on Nicolet DX and Kratos MS 80 RFA spectrometers,
respectively. Optical rotation was measured on JASCO DIP-370
polarimeter. Melting points were taken on an Electrothermal apparatus
and are uncorrected. Thin-layer chromatograms were made on silica
gel. The purification protocols for the TEM-12 and Q908R3 â-lacta-
mases have been described previously. Kinetic measurements were
carried out on a Hewlett-Packard 452 diodearray instrument. All
enzyme assays were performed in 100 mM sodium phosphate buffer,
pH 7.0. (5R,2R)-1-N-Benzyl-5-[(methoxycarbonyl)methyl]pyrrolidine-
2-carboxylic acid tert-butyl ester (7)4 and (1R,3S)-cyclopentanedicar-
boxylic acid 1-methyl ester (13)5 were synthesized according to the
literature methods. Modeling was performed according to the meth-
odology reported previously with AMBER force field.6
30.0, 38.6, 54.9, 60.6, 80.5, 81.2, 153.6, 172.3, 176.3; [R]22 +98° (c
D
0.617, CH2Cl2); EI MS 330 (M+, 1%).
(5R,2R)-1-N-(tert-Butoxycarbonyl)-5-[[(p-nitrophenoxy)carbonyl]-
methyl]pyrrolidine-2-carboxylic Acid tert-Butyl Ester (11). p-
Nitrophenol (90 mg, 0.65 mmol), 1,3-dicyclohexylcarbodiimide (128
mg, 0.62 mmol) and 1-hydroxybenzotriazole monohydrate (95 mg, 0.62
mmol), were added to the solution of 10 (209 mg, 0.63 mmol) in ethyl
acetate (20 mL). The reaction mixture was stirred for 24 h at room
temperature. The mixture was filtered, and the solvent was removed
by evaporation. The residue was redissolved in ethyl acetate (20 mL)
and filtered again. The solvent was removed, and the residue was
purified by silica gel column chromatography (hexane/ethyl acetate,
5/2) to afford the title compound as a yellow oil (185 mg; yield, 65%):
(5R,2R)-5-[(Methoxycarbonyl)methyl]pyrrolidine-2-carboxylic Acid
tert-Butyl Ester (8). A suspension of 35 mg of 5% Pd/C in methanol
1
Rf 0.51 (hexane/ethyl acetate, 5/2); H NMR (CDCl3) δ 1.38 (9H, s,
(2 mL) was stirred under an atmosphere of hydrogen for 10 h.
A
t-Bu), 1.41 (9H, s, t-Bu), 1.81-1.90 (1H, m, C3 or C4 methylene),
1.81-2.02 (1H, m, C3 or C4 methylene), 2.17-2.30 (2H, broad m,
C3 or C4 methylene), 2.69 (1H, dd, J ) 8.5 and 16 Hz, C6 methylene),
3.40 (1H, dd, J ) 5 and 16 Hz, -CH2CO2R), 4.19 (1H, m, C5 methine),
4.40 (1H, m, C2 methine), 7.32 (2H, d, J ) 9 Hz, aromatic), 8.25 (2H,
d, J ) 9 Hz, aromatic); 13C NMR (CDCl3) δ 28.0, 28.3, 28.8, 30.1,
39.0, 55.0, 60.7, 80.4, 81.2, 122.5, 125.1, 145.3, 153.5, 155.5, 169.3,
172.1; EI MS 329 (M - p-nitrophenol, 1%).
solution of 7 (110 mg, 0.33 mmol) in methanol (5 mL) was added to
the suspension, and the mixture was stirred for 15 h. The mixture was
filtered, and the filtrate was evaporated in Vacuo to give a solid residue.
(1) Lobkovsky, E.; Moews, P. C.; Liu, H.; Zhao, H.; Fre`re, J. M.; Knox,
J. R. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11257.
(2) Zafaralla, G.; Manavathu, E. K.; Lerner, S. A.; Mobashery, S.
Biochemistry 1992, 31, 3847.
(5R,2R)-5-[[(p-Nitrophenoxy)carbonyl]methyl]pyrrolidine-2-car-
boxylic Acid (2). Compound 11 (100 mg, 0.22 mmol) was dissolved
in a mixture of 5 mL of freshly distilled trifluoroacetic acid and 1 mL
of anisole. The reaction mixture was stirred for 8 min at ice-water
temperature, after which trifluoroacetic acid was removed in Vacuo.
Both water and ethyl acetate, 2 mL each, were added to the resulting
brown oil. The aqueous fraction was lyophilized resulting in the title
(3) Ross G. W. Methods Enzymol. 1975, 43, 678.
(4) Petersen, S. P.; Fels, G.; Rapoport, H. J. Am. Chem. Soc. 1984, 106,
4539.
(5) Cheˆnevert, R.; Lavoie, M.; Courchesne, G.; Martin, R. Chem. Lett.
1994, 93.
(6) (a) Massova, I.; Fridman, R.; Mobashery, S. J. Mol. Mod. 1997, 3,
17. (b) Bulychev, A.; O’Brien, M. E.; Massova, I.; Teng, M.; Gibson, T.
A.; Miller, M. J.; Mobashery, S. J. Am. Chem. Soc. 1995, 117, 5938.

Versatile â-Lactam Hydrolase ActiVity
J. Am. Chem. Soc., Vol. 119, No. 33, 1997 7621
1
compound as a yellow solid (41 mg; yield, 60%): mp 94 °C dec; H
NMR (D2O) δ 1.64-1.80 (1H, m, C3 or C4 methylene), 2.11-2.32
(3H, broad m, C3 or C4 methylene), 3.17 (2H, dd, J ) 4 Hz, -CH2-
CO2R), 4.02 (1H, m, C5 methine), 4.27 (1H, m, C2 methine), 7.26
(2H, d, J ) 9 Hz, aromatic), 8.18 (2H, d, J ) 9 Hz, aromatic); 13C
NMR (D2O) δ 35.7, 40.3, 41.3, 48.5, 69.3, 85.3, 135.3, 145.6, 154.6,
carboxylic Acid (3). p-Nitrophenol (115 mg, 0.83 mmol), 1,3-
dicyclohexylcarbodiimide (164 mg, 0.8 mmol), and 1-hydroxybenzo-
triazole monohydrate (121 mg, 0.8 mmol) were added to a solution of
15 (139 mg, 0.81 mmol) in ethyl acetate (25 mL). The reaction mixture
was stirred for 24 h at room temperature. The solution was filtered,
and the filtrate was concentrated to dryness in Vacuo. The residue was
redissolved in ethyl acetate, and the solution was filtered again. The
solvent was evaporated, and the residue was purified by silica gel
column chromatography (hexane/ethyl acetate, 1/1) to afford the title
compound as a yellowish solid (70 mg; yield, 30%): Rf 0.48 (hexane/
ethyl acetate, 2/3); mp 87 °C dec; 1H NMR (CDCl3) δ 1.41-1.52 (1H,
m, C2 methylene), 1.59-1.68 (1H, m, C2 methylene), 1.97-2.13 (4H,
broad m, C4 and C5 methylene), 2.33-2.43 (1H, m, C3 methine), 2.49
(2H, d, J ) 3 Hz, CH2CO2R), 3.08-3.13 (1H, m, C1 methine), 7.27
(2H, d, J ) 6 Hz, aromatic proton), 8.27 (2H, d, J ) 6 Hz, aromatic
proton), 10.84 (1H, broad s, carboxylic acid); 13C NMR (CDCl3) δ
29.4, 32.6, 35.2, 37.5, 40.2, 45.9, 119.1, 125.5, 141.5, 127.2, 161.2,
168.3, 183.0; [R]25 +134° (c 0.743, H2O); FAB MS 295 (M, 58%).
D
(1R,3S)-3-[[(Benzyloxy)carbonyl]methyl]cyclopentanecar-
boxylic Acid Methyl Ester (14). The general procedure for one-carbon
homologation of the carboxylic acid, as well as the issue of configu-
ration retention are described elsewhere.7 Oxalyl chloride (3.6 mL,
20 mmol) was added under stirring dropwise to the solution of 13 (730
mg, 4.3 mmol) in dry CH2Cl2 (10 mL) under an atmosphere of nitrogen.
A catalytic amount of DMF (one drop) was added, and the reaction
mixture was stirred at room temperature for 2.5 h under nitrogen. The
solvent and excess oxalyl chloride were removed in Vacuo to give the
crude acyl chloride as a brown oil. The acyl chloride was attached to
high vacuum for 6 h and was taken to the next step without any
purification: 1H NMR (CDCl3) δ 1.96-2.45 (6H, unresolved m, C2,
C4 and C5 methylene), 2.88 (1H, m, C3 methine), 3.29 (1H, m, C1
methine), 3.70 (3H, s, methyl).
173.3; [R]23 +96° (c 0.914, H2O); CI MS 172 (M + H - p-
D
nitrophenol, 1%).
Turnover Kinetics. Compounds 2 and 3 experienced some residual
hydrolysis in neutral buffered solutions. Hence, all rates for enzymatic
turnover of these compounds were obtained by subtracting the
background hydrolysis rate from the corresponding rates of the enzyme-
catalyzed reactions. The kcat and Km parameters for turnover of
compounds 2 and 3 by the TEM-1 â-lactamase were determined from
Lineweaver-Burk plots. The standard assay conditions were as
follows. Portions of the stock solutions of compounds in dioxane were
added to the 1-mL assay containing 100 mM phosphate buffer pH 7.0
and 33 nM TEM-1 enzyme to give final concentrations of 1.0-5.0
mM for 2, and 0.5-2.0 mM for 3. The initial rates for hydrolyses
were monitored at 455 nm (∆ꢀ455 1114 M-1 cm-1 and 1121 M-1 cm-1
for compounds 2 and 3, respectively). The enzymes are fully active
in the presence of 10% dioxane as reported earlier.6b The kcat and Km
values for turnover of compound 2 by the Q908R enzyme were
determined similarly. Portions of the stock solution of compound 2 in
dioxane were added to the 1-mL assay containing 100 mM sodium
phosphate buffer, pH 7.0, and 80 nM of the Q908R enzyme to give
final substrate concentrations of 1-10 mM, prior to monitoring of the
rates of hydrolysis.
(Trimethylsilyl)diazomethane (4.5 mL of 2 M solution in hexanes)
was added dropwise to a stirred solution of the acyl chloride in a 1:1
mixture of anhydrous THF and acetonitrile (20 mL total) under an
atmosphere of dry nitrogen at ice-water temperature. The reaction
mixture was stirred for 5 h, after which another portion of the
(trimethylsilyl)diazomethane solution was added (4 mL), and the
mixture was allowed to progress for another 5 h. The volatiles were
removed in Vacuo to give the crude diazo ketone as a brown oil. After
the crude product was kept under high vacuum for 6 h, it was dissolved
in a 1:1 mixture of benzyl alcohol and 2,4,6-trimethylpyridine (4 mL
total). The solution was stirred under an atmosphere of dry nitrogen
at 180-185 °C for 7 min. The crude product was purified by silica
gel column chromatography (benzene/hexane/diethyl ether, 1/1/0.2) to
afford the title compound as a colorless oil (801 mg; yield, 68%): Rf
0.46 (benzene/hexane/diethyl ether, 1/1/0.25); 1H NMR (CDCl3) δ
1.25-1.54 (2H, broad m, C2 methylene), 1.82-1.95 (3H, broad m,
C4 or C5 methylene), 2.19-2.23 (1H, m, C4 or C5 methylene), 2.25-
2.39 (1H, m, C3 methine), 2.42 (2H, d, J ) 8 Hz, -CH2CO2R), 2.73-
2.85 (1H, m, C1 methine), 3.64 (3H, s, methyl), 5.10 (2H, s, -CH2Ph),
7.33 (5H, m, aromatic); 13C NMR (CDCl3) δ 28.8, 29.7, 31.9, 36.4,
36.7, 39.8, 43.3, 51.6, 66.1, 128.1, 128.5, 136.0, 172.7, 176.6; [R]24
+61° (c 1.012, ethyl acetate); EI MS 276 (M, 1%).
Inactivation Experiments. A solution of 3 in dioxane was added
to a solution of the Q908R â-lactamase (0.2 µM) in 100 mM sodium
phosphate buffer, pH 7.0 (final dioxane concentration of 10%), in a
total volume of 50 µL, to give final concentrations of 3 in the range of
0.2-3.0 mM. Aliquots (10 µL) of the inactivation mixture were diluted
into the 1-mL assay mixture containing 0.1 mM cephaloridine in the
same buffer. The initial rate of hydrolysis of cephaloridine was
monitored at 295 nm (∆ꢀ295 cephaloridine 1000 M-1 cm-1).
D
(1S,3S)-3-(Carboxymethyl)cyclopentanecarboxylic Acid (15). A
solution of NaOH (16 mL of 1 M) was added to a solution of 14 (200
mg, 0.73 mmol) in dioxane (16 mL), subsequent to which, the reaction
mixture was stirred for 24 h at room temperature. The solvent and
low boiling components were removed in Vacuo to give the crude
intermediate in the form of a viscous oil. The oil was fractionated
between water and ethyl acetate. The aqueous fractions were concen-
trated in Vacuo to result in a white crystalline solid. The product was
dissolved in 1 M HCl (1.5 mL) and was heated at 180 °C for 6 h in a
sealed tube, as described by Berson and Reynolds-Warnhoff.8 The
product was extracted into ethyl acetate (2×, 40 mL each). The
combined organic fraction was evaporated in Vacuo to give the title
The enzyme inactivation process was accompanied by the rapid
release (“burst”) of p-nitrophenolate, a process which was dependent
on the concentration of the enzyme that was being inactivated.
portion of the Q908R â-lactamase (4.5 or 12.7 µM final concentration)
was added to 1-mL assay containing compound 2 (1 mM) in 100 mM
sodium phosphate buffer, pH 7.0. The change of the absorbance due
to release of p-nitrophenolate as a function of time was monitored at
455 nm. The rates of release of p-nitrophenolate were corrected for
the nonenzymic hydrolysis of 2.
A
1
compound as white crystals (110 mg; yield, 88%): mp 131 °C; H
NMR (D2O) δ 1.34-1.43 (1H, m, C2 methylene), 1.49-1.55 (1H, m,
C2 methylene), 1.89-1.99 (3H, broad m, C4 or C5 methylene), 2.19-
2.26 (1H, m, C4 or C5 methylene), 2.28-2.35 (1H, m, C3 methine),
2.44 (2H, d, J ) 4 Hz, -CH2CO2H), 2.82-2.89 (1H, m, C2 methine);
Product Analysis. Compounds 2 and 3 were hydrolyzed by the
TEM-1 â-lactamase. The protein was separated from the low molecular
weight products by ultrafiltration in an Amicon device. The products
were analyzed by HPLC, and were compared to the authentic standards
for each case. The conditions were as follows: Waters C-4 1.0 × 25
cm, 5-95% linear acetonitrile gradient, 0.1% TFA, over 40 min, 1
mL/min, 205 and 405 nm; p-nitrophenolate, tR ) 38 min (205 nm or
405 nm); (5R,2R)-5-(carboxymethyl)pyrrolidine-2-carboxylic acid, tR
) 14 min (205 nm); (1S,3S)-3-(carboxymethyl)cyclopentanecarboxylic
acid (15), tR ) 31 min (205 nm); (1S,3S)-3-[[(p-nitrophenoxy)carbonyl]-
methyl]cyclopentanecarboxylic acid (3), tR ) 45 min (Waters C-4 1.0
× 25 cm, 5-95% linear acetonitrile gradient, 0.1% TFA, over 40 min;
95% acetonitrile, 0.1% TFA, over 10 min, 1 mL/min, 205 or 405 nm).
13C NMR (D2O) δ 28.8, 31.8, 36.2, 36.4, 39.4, 43.3; [R]23 +84° (c
D
1.006, H2O); EI MS 172 (M+, 1%).9
(1S,3S)-3-[[(p-Nitrophenoxy)carbonyl]methyl]cyclopentane-
(7) Aoyama, T.; Shioiri, T. Chem. Pharm. Bull. 1981, 29, 3249.
(8) Berson, J. A.; Reynolds-Warnhoff, P. J. Am. Chem. Soc. 1964, 86,
595.
(9) The optical rotation for (1S,3S)-3-(carboxymethyl)cyclopentanecar-
boxylic acid (15) was [R]23D ) +84° (c ) 1.006, H2O) and that for (1R,3S)-
3-(carboxymethyl)cyclopentanecarboxylic acid was [R]23 ) +38° under
D
the same conditions. Furthermore, (1R,3S)-3-(carboxymethyl)cyclopentan-
ecarboxylic acid showed NOE of 2% between C3 and C1 hydrogens,
whereas (1S,3S)-3-(carboxymethyl)cyclopentanecarboxylic acid (15) did not
give any NOE.

7622 J. Am. Chem. Soc., Vol. 119, No. 33, 1997
BulycheV et al.
Scheme 1
approach to the acyl carbonyl from the â-face.16 To test this
possibility, compounds 2 and 3 were designed. Both compounds
are surrogates of a minimal “â-lactam” molecule. Compound
2 was expected to acylate the active-site serine of the class C
â-lactamase, followed by deacylation (2 f 4 f 5), should the
Scheme 2
ring nitrogen promote the deacylation step. On the other hand,
compound 3 was anticipated solely to acylate the active site,
and not having the requisite amine in its structure, the
intermediate should resist deacylation. Both compounds would
be expected to serve as substrates for the class A TEM-1
â-lactamase, since the approach of water in this enzyme is from
the R-face, but also the process is not substrate-assisted, as
suggested here for the class C enzymes.
Compounds 2 and 3 were synthesized in multiple steps
according to the Schemes 1 and 2. Compounds 7 and 13 were
the key starting materials for the syntheses of the target
compounds, which were prepared from D-Glu (6)4 and nor-
bornylene (12)5 according to literature methods, respectively.
As expected, compounds 2 and 3 were both substrates for
the class A TEM-1 â-lactamase (2, Km ) 3.52 ( 0.35 mM, kcat
) 1.1 ( 0.2 s-1, kcat/Km ) 312 ( 65 M-1 s-1; 3, Km ) 0.92 (
0.08 mM, kcat ) 0.17 ( 0.03 s-1, kcat/Km ) 185 ( 36 M-1
s-1). On the other hand, compound 2 was a substrate for the
Results and Discussion
A molecular probe, 6R-(hydoxymethyl)penicillanic acid (1),
was designed in our earlier work in a computer-aided process
with the help of the crystal structure for the Escherichia coli
TEM-1 â-lactamase, a prototypic class A enzyme. This
molecule was designed to prevent the approach of the presumed
hydrolytic water from the R-face of the acyl-enzyme intermedi-
ate.10 The compound acylated the enzyme readily, but resisted
deacylation, as was expected. The crystal structure for the acyl-
enzyme intermediate for 1, the only acyl-enzyme intermediate
for turnover of a substrate by a native class A â-lactamase,11
supported the design paradigms, indicating that the approach
of the hydrolytic water is indeed from the R-face, and is
promoted by Glu-166 as a general base.12
class C Q908R17 â-lactamase (Km ) 5.56 ( 0.23 mM, kcat
)
0.050 ( 0.006 s-1, kcat/Km ) 9.0 ( 1.1 M-1 s-1), but compound
3 served as an inactivator for the enzyme (KI ) 6.2 ( 0.7 mM,
kinact ) 0.21 ( 0.06 s-1, kinact/KI ) 34 ( 5 M-1 s-1).18
Compound 3 acylated the enzyme efficiently, as evidenced by
the rapid release of p-nitrophenolate, in a process which was
directly dependent on concentration of the enzyme (Figure 1),
(16) Deprotonated Tyr-150 has been suggested as the general base for
deacylation of the acyl-enzyme intermediate in class C enzymes by one
group (Dubus, A.; Ledent, P.; Lamotte-Brasseur, J.; Fre`re, J. M. Proteins:
Struct., Funct., Genet. 1996, 25, 473), whereas the results of another group
indicated that this is not so (Dubus, A.; Normark, S.; Kania, M.; Page, M.
G. P. Biochemistry 1994, 33, 8577). The ring nitrogen, as will be outlined
in the text, plays an important role in the deacylation step.
(17) The P99 and Q908R â-lactamases are different only in four amino
acids at sites remote from the active site. The two enzymes are believed to
be virtually identical in both structure and mechanism.
(18) The enzyme activity gradually recovered from inhibition over several
hours (kre ) 5.7 × 10-5 s-1). This rate of deacylation (i.e., recovery of
activity) is comparable to those for PBPs acylated by the â-lactam
antibiotics, which are devoid of the catalytic machinery for deacylation
(typically in the range of 10-4 to 10-6 s-1): Wilkin, J. M.; Jamin, M.;
Joris, B.; Fre`re, J. M. Biochem. J. 1993, 293, 195. Wilkin, J. M.; Dubus,
A.; Joris, B.; Fre`re, J. M. Biochem. J. 1994, 301, 477.
Interestingly, there is no counterpart to Glu-166 in class C
â-lactamases.1 Earlier work from our laboratory13 and informa-
tion from the crystal structures for the Citrobacter freundii,14
and for the Enterobacter cloacae P99 â-lactamases,1,15 both class
C â-lactamases, had suggested that the approach of the
hydrolytic water may be from the â-face of the acyl-enzyme
species for these enzymes. It occurred to us that if this were
the case, the formerly â-lactam nitrogen, now a secondary amine
at the acyl-enzyme intermediate stage, is ideally positioned to
serve as a general base in promoting the water molecule for
(10) Miyashita, K.; Massova, I.; Taibi, P.; Mobashery, S. J. Am. Chem.
Soc. 1995, 117, 11055.
(11) Maveyraud, L; Massova, I.; Birck, C.; Miyashita, K.; Samama, J.
P.; Mobashery, S. J. Am. Chem. Soc. 1996, 118, 7435.
(12) Our work with compound 1 complemented earlier mutagenesis
results (Adachi, H.; Ohta, T.; Matsuzawa, H. J. Biol. Chem. 1991, 266,
3186. Escobar, W. A.; Tan, A. K.; Fink, A. L. Biochemistry 1991, 30, 10783.
Delaire, M.; Lenfant, F.; Labia, R.; Masson, J. M. Prot. Eng. 1991, 4, 805),
which support these conclusions.
(13) Bulychev, A.; Massova, I.; Lerner, S. A.; Mobashery, S. J. Am.
Chem. Soc. 1995, 117, 4797.
(14) Oefner, C.; Darcy, A.; Daly, J. J.; Gubernator, K.; Charnas, R. L.;
Heinze, I.; Hubschwerlen, C.; Winkler, F. K. Nature 1990, 343, 284.
(15) Lobkovsky, E.; Billings, E. M.; Moews, P. C.; Rahil, J.; Pratt, R.
F.; Knox, J. R. Biochemistry 1994, 33, 6762.
(19) The pH-dependence plots for kcat and kcat/Km for catalysis by the
class C enzymes are bell shaped and indicate the potential involvement of
a group at the basic limb of catalysis which titrates with a pKa value of
∼10: Page M. I.; Vilanova, B.; Layland, N. J. J. Am. Chem. Soc. 1995,
117, 12092. Although the authors interpreted this finding as probably due
to Tyr-150, we believe that if the decline in the catalytic ability at the basic
limb of the pH profile is due to a titratable function, it is also consistent
with the ring amine as such a residue. The alternative is that both the ring
amine and the side chain of Tyr-150 are titrating simultaneously. Here are
some typical pKa values relevant to this discussion: 10.7 ( 0.2 for proline,
8.8 ( 0.2 for thiazolidine, and 11.3 ( 0.2 for pyrrolidine. The dependence
of pH of reactions with 2 and 3 could not be studied because of the instability
of the p-nitrophenyl esters at both acidic and basic conditions.

Versatile â-Lactam Hydrolase ActiVity
J. Am. Chem. Soc., Vol. 119, No. 33, 1997 7623
base. It is worthy of a note that the distance between the side-
chain oxygen of Tyr-150 and the ring nitrogen of the acyl-
enzyme intermediate is in excess of 5 Å, precluding any direct
interaction between the two, although they would both be
influenced by one another in an electrostatic sense.
We note that Tyr-150, ring nitrogen, and the substrate
carboxylate are all within a hydrogen-bonding distance to an
active-site water molecule. Comparative work of Waley with
the cephalosporin lactone 16 and typical â-lactam substrates
had indicated some influence by the carboxylate of the â-lactam
substrates on enzymic hydrolyses.21 This information in
conjunction with results of mutagenesis at position 150 and our
findings reported herein indicate a collective influence by these
functionalities on the deacylation step of the hydrolytic mech-
anism. When the class C enzyme is acylated by 3, its catalytic
machinery is impaired sufficiently such that enzyme inactivation
results. The activity returns over several hours, suggesting that
deacylation occurs, albeit sluggishly.18 Similarly, the results
of Waley indicated that the absence of the substrate carboxylate
(as in lactone 16) affected hydrolysis.21 The work of Pratt and
colleagues with non-â-lactam peptide and depsipeptide sub-
strates for class C â-lactamases is interesting in that the portion
of the substrate corresponding to the substrate ring is released
in the course of turnover.22 These are substrates which either
are hydrolyzed by entirely different mechanism(s), or they rely
solely on the functionalities of the enzyme (e.g., Tyr-150) to
facilitate the slow deacylation. In summary, we believe that
activation of the hydrolytic water molecule in the active site of
class C enzymes would potentially rely on the functions of the
substrate carboxylate, ring nitrogen and Tyr-150; the ring
nitrogen would potentially serve as the active-site general base,
which activates the water molecule for the deacylation step, or
it affects the electrostatic properties of the active site so
significantly that its absence from the active site impairs
catalysis.
Figure 1. Release of p-nitrophenol due to modification of the active
site of the Q908R â-lactamase (at specified concentrations) by
compound 3. The measurements were made in 100 mM sodium
phosphate, pH 7.0, with concentration of compound 3 fixed at 6.7 mM
in each case. The extrapolated points for the “burst size” are proportional
to the respective enzyme concentrations.
and the acylated enzyme resisted deacylation. These observa-
tions are consistent with our proposal that the ring amine in
â-lactam antibiotics would play an important role in the
deacylation step.19 Hence, in contrast to class A â-lactamases,
substrate-assisted catalysis would appear to be operating for class
C enzymes.20
We were prompted to prepare and study compounds 2 and 3
on the basis of our initial notion that the ring nitrogen might
serve as the general base in promoting the deacylation step.
However, we are aware that the electrostatic contribution of
the ring nitrogen to the stabilization of the transition state for
deacylation may also be a factor. That is to say, if the ring
nitrogen is not the general base for promotion of water in the
hydrolytic process, its electrostatic contribution to the reaction
energy profile is still quite significant for class C â-lactamases,
but not so for class A â-lactamases. In essence, the collective
nature of the active-site functionalities may be considered
perturbed in the absence of the ring nitrogen, hence affecting
the catalytic ability of the enzyme to undergo deacylation after
modification of the active site by compound 3. Deprotonated
Tyr-150 has been suggested as the general base for deacylation
of the acyl-enzyme intermediate in class C enzymes by one
group, whereas the results of another group showed some
significant effects on the catalytic parameters for turnover of
substrates, but were inconsistent with this conclusion.16 There
are no crystal structures for a typical acyl-enzyme intermediate
available for a class C â-lactamases, so we have generated an
energy-minimized structure for the acyl-enzyme intermediate
of cephalothin, a first-generation cephalosporin, in the active
site of the crystal structure for the class C enzyme from E.
cloacae P99 (Figure 2). We believe that because of the close
interactions of the side chains of Lys-315 and Lys-67 with that
of Tyr-150, it is likely that the Tyr-150 side chain may exist in
its ionized phenolate form. The phenolate contribution to the
electrostatic nature of the surface of the active site should be
significant, which is consistent with the results of mutagenesis
experiments at this position, indicating this residue to be indeed
important for the enzyme, even if it may not be the general
A recent parsimony analysis of â-lactamases indicated that
divergence of classes A and C of these enzymes from a common
ancestor was an early event in their diversification of function.23
Bacteria have been in existence for over 3.5 billion years,24 and
the chances are that PBPs have been serving their biosynthetic
activities from the early beginnings of bacterial life. The advent
of antibiotics, such as â-lactams, by microorganisms took place
in order to give the producing organisms an advantage over
the nonproducing bacteria. The biosynthetic development of
antibacterials is an old one. Indeed, it is accepted that the
resistance mechanisms to antibacterials arose in response to the
challenge by antibiotic-producing organisms, well predating the
selection pressure due to clinical use of antibiotics in the modern
era.25 Reconstruction of the biochemical evolutionary paths for
(21) Waley, S. G. In The Chemistry of â-Lactams; Page, M. I., Ed.;
Blackie Academic: London, 1992; pp 216-217.
(22) Pazhanisamy, S.; Govardhan, C. P.; Pratt, R. F. Biochemistry 1989,
28, 6863. Murphy, B. P.; Pratt, R. F. Biochemistry 1991, 30, 3640. Xu, Y.;
Soto, G.; Hirsch, K. R.; Pratt, R. F. Biochemistry 1996, 35, 3595. Adediran,
S. A.; Deraniyagala, S. A.; Pratt, R. F. Biochemistry 1996, 35, 3604.
(23) Bush, K.; Jacoby, G. A.; Medeiros, A. A. Antimicrob. Agents
Chemother. 1995, 39, 1211.
(20) We have observed that under the conditions that the kinetic
experiments were carried out (100 mM phosphate buffer pH 7.0, room
temperature), compound 2 underwent nonenzymic hydrolysis approximately
5-fold faster than did 3. It is plausible that the ring nitrogen may also be
involved in enhancing this solvent-mediated hydrolysis.
(24) Holland, H. D. Science 1997, 275, 38.
(25) Abraham, E. P.; Chain, E. Nature 1940, 146, 837. Massova, I.;
Mobashery, S. Acc. Chem. Res. 1997, 30, 162.

7624 J. Am. Chem. Soc., Vol. 119, No. 33, 1997
BulycheV et al.
a
b
Figure 2. (A) Stereoview of the energy-minimized structure for the acyl-enzyme intermediate for cephalothin in the active site of the class C
â-lactamase from E. cloacae P99. Cephalothin and the hydrolytic water molecule are shown in bold. Because of the fleeting existence of the
acyl-enzyme species, the acetoxy group at the C3 position is retained; the expulsion of this group is known to take place once the product of
hydrolysis is released into solution. (B) A different perspective, showing the proximity of the side chain of Tyr-150, the substrate ring nitrogen and
carboxylate to the hydrolytic water, all of which are within a hydrogen-bonding distance.
such a highly evolved system presents difficulties since any such
analysis has to be carried out on the basis of our knowledge of
the properties of the extant modern descendants. However,
â-lactamases present a special case for such analyses since by
now over 200 members of this family of enzymes have been
identified,23 and that several crystal structures for representative
enzymes are available. Analysis of the details of mechanisms
of â-lactamases would be revealing in understanding the
incremental evolutionary steps which would lead to the modern
forms of these enzymes. Such an undertaking is attempted in
this manuscript.
The suggestion that a certain ancestral bacterial biosynthetic
enzyme (e.g., a PBP) may have given rise to the resistance
enzymes was put on firm ground on the basis of the observation
of the general conservation of the three-dimensional topology
of the DD-peptidase/transpeptidase from Streptomyces R61, a
PBP, with the crystal structures of â-lactamases.26 We have
used the mechanistic features of â-lactamases as they pertain
to the deacylation step as a tool in gaining insight into the
evolutionary pathways for these related enzymes. Nature has
selected two distinct pathways for deacylation of the acyl-
enzyme intermediates in catalysis by classes A and C of
â-lactamases. The approach of water to the acyl carbonyl is
from the R-face for the class A â-lactamases, whereas for the
class C enzymes it is from the â-face. An interesting aspect of
the structures for classes A and C of â-lactamases and the PBPs
is that the general scaffolding for the active-site functionalities
is remarkably preserved. In this vein, the information from the
crystal structure for DD-peptidase/transpeptidase from Strep-
tomyces R61 is informative. We note that the active site of
this PBP contains two important crystallographic water mol-
ecules (Figure 3). When the structure of this enzyme is
superimposed on those of the class A â-lactamases (including
the TEM-1 enzyme), the position of the hydrolytic water of the
class A enzymes is identical to that of Wat-1 of DD-peptidase/
transpeptidase from Streptomyces R61 (Figure 3). Hence, in
the course of evolution of the class A enzymes, the ancestral
enzyme underwent restructuring near the active site to make
available Glu-166 for activating Wat-1 for hydrolysis, a water
molecule which already existed in the active site of the PBP.
On the other hand, in evolution of the class C enzymes nature
took a different ploy. Instead of relying solely on a basic
function in the active site, as was the case for the class A
enzymes, these enzymes exploited the ring amine of the acyl-
enzyme species in facilitating the deacylation step. We hasten
to add that Tyr-159 of DD-peptidase/transpeptidase (which is
devoid of deacylation machinery) corresponds spatially to Tyr-
150 of the class C â-lactamases, so Tyr-150 was not put in
place to facilitate the deacylation step in class C enzymes. The
water molecule on the â-face of the acyl-enzyme species (Wat-
2) can serve as the potential hydrolytic water molecule (Figure
3). This water molecule is positioned less than 0.5 Å away
from a suitable interaction with the ring amine of the acyl-
enzyme species in the crystal structure of DD-peptidase/
transpeptidase (Figure 3). Therefore, nature would require the
restructuring of the environment around this water molecule to
permit its approach to the ring amine and the carbonyl of the
(26) Kelly, J. A.; Dideberg, O.; Charlier, P.; Wery, J. P.; Libert, M.;
Moews, P. C.; Knox, J. R.; Duez, C.; Fraipont, C.; Joris, B.; Dusart, J.;
Fre`re, J. M.; Ghuysen, J. M. Science 1986, 231, 1429.

Versatile â-Lactam Hydrolase ActiVity
J. Am. Chem. Soc., Vol. 119, No. 33, 1997 7625
Figure 3. The active site of DD-peptidase/transpeptidase from Streptomyces R61 modified by cephalothin (see below for schematic of the structure),
shown as the water-accessible surface from the crystal structure (Kuzin, A. P.; Liu, H.; Kelly, J. A.; Knox, J. R. Biochemistry 1995, 34, 9532). Two
active-site water molecules are shown as spheres at 2 and 5 o’clock positions. Because of the longevity of the acyl-enzyme species, the acetoxy
group appended to the C3 methyl is expelled.
acyl-enzyme species during its metamorphosis to a class C
â-lactamase.
classes A and C of â-lactamases from an ancestral PBP was
likely to have been two independent events.27
On the basis of conservation of structural topology, Lobk-
ovsky et al. suggested that evolution of these enzymes was a
sequential one;1 that is to say that the PBP gave rise to the larger
class C enzymes, which in turn underwent change to become
class A enzymes. Our findings reported in this manuscript do
not support this conclusion. A linear evolution of one â-lac-
tamase from another intuitively becomes impossible, since the
existing deacylation machinery in the first enzyme would have
to be dismantled first, and then another should be put into place,
if the linear evolution had taken place. We suggest here that
in light of the mechanistic evidence presented, evolution of
Note Added in Proof: Subsequent to submission of this
manuscript, a structural survey of two â-lactamases and one
penicillin-binding protein by Knox et al. has appeared in the
literature (Knox, J. R.; Moews, P. C.; Fre`re, J. M. Chemistry &
Biology 1996, 3, 937), which is relevant to the question of
independent evolution for the classes A and C of â-lactamases.
Acknowledgment. This work was supported by the National
Institutes of Health. I.M. is the recipient of the Rumble and
Heller Predoctoral Fellowships. We are indebted to Professors
Judith Kelly and James Knox for supplying us the coordinates
for the of DD-peptidase/transpeptidase from Streptomyces R61
prior to its availability from the Protein Data Bank.
(27) We have carried out a multiple amino acid sequence alignment of
73 distinct â-lactamases of all four classes with 80 known PBPs. The
alignment indicated that PBPs diversified into six distinct clusters. As
branching points from the PBPs, â-lactamases were sequestered. For
example, all class A â-lactamases were more closely related to one group
of PBPs, and similarly all class C â-lactamases were more closely related
to a different group of PBPs. This observation further supports our notion
for a nonlinear process for the evolution of â-lactamases from PBPs.
(Massova and Mobashery, unpublished results).
Supporting Information Available: The 1H and 13C NMR
spectra for compounds 2 and 3 (4 pages). See any current
masthead page for ordering and Internet access instructions.
JA963708F