Sompop Bencharit1,2 and Matthew R. Redinbo1,3
1Department of Chemistry, University of North Carolina at Chapel
Hill, Chapel Hill, North Carolina
2School of Dentistry, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina
3Department of Biochemistry & Biophysics and the Lineberger
Comprehensive Cancer Center, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina
Mammalian carboxylesterases are promiscuous enzymes responsible for processing
a wide variety of drugs and xenobiotics (Redinbo et al.,
carboxylesterases metabolize and detoxify the dangerous narcotics heroin and
cocaine, and the potent chemical weapon agents Sarin, Soman and Tabun.
We have published the first three crystallographical studies of this
distinctive group of enzymes and proposed detailed structure-function
findings have implications for drug design, the treatment of cocaine and heroin
abuse, and victims of chemical warfare or terrorism. Beam Lines 7-1, 9-1 and
9-2 at Stanford Synchrotron Radiation Laboratory (SSRL) played a significant
role in the data collection necessary to assemble these biological insights.
proposed mechanism for the activation of irinotecan (CPT-11) by rCE.
CPT-11 (orange) enters from the top of the catalytic gorge and fits well into
the active site (catalytic Ser-221 and Glu-353 in green). After cleavage, the
alcohol product (SN-38; magenta) leaves via the catalytic gorge, while the acyl
product (4PP; purple) moves past the gate residues (cyan) and docks adjacent to
the regulatory domain (red) on the surface of the molecule.
First, the structure of a mammalian carboxylesterase, that of rabbit
liver carboxylesterase (rCE), reveals the activation mechanism of the
camptothecin anticancer drug, irinotecan (CPT-11) (Bencharit et al., 2002). Using
the position of a bound product on the surface of the enzyme, we have proposed
that rCE utilizes a "side door" secondary product exit pore to facilitate the
efficient processing of irinotecan (Fig 1).
Second, our work on human carboxylesterase 1(hCE1), the human homolog of rCE,
are the first structures of a human enzyme bound to cocaine and heroin
analogues (Fig 2a-b) (Bencharit et al., 2003a). The hCE1 active
site contains both specific and promiscuous compartments, which enable the
enzyme to act on structurally distinct chemicals. The position of bound
homatropine, a cocaine
analogue, and bound naloxone, a heroin analogue, reveal how hCE1 utilizes its
large flexible pocket promiscuously and its small rigid pocket specifically.
These crystal structures together with the results from atomic force microscopy
provide the first evidence of hCE1's trimer-hexamer equilibrium.
a. The hCE1 trimer as observed in the homatropine complex;
homatropine molecules bound at the active sites are gold. Homatropines bound at
the Z site, between the Ω loops, are gold and purple.
b. Two orientations of naloxone methiodide (green and orange) bound within
the hCE1 catalytic pocket. The two binding modes of this heroin analog are
related by an opposite two-fold rotation about the vertical axis. The molecular
surfaces of the catalytic binding pocket corresponding to the two orientations
of the naloxone ligand are rendered in transparent green and orange, and
demonstrate the induced fit property of the substrate-binding pocket.
surface ligand-binding site regulates the trimer-hexamer equilibrium and allows
each monomer to bind cocaine and heroin analogs simultaneously.
resolution simulated annealing omit maps (contoured at 3.0s) showing the
electron density for tacrine bound at the active site of hCE1. Multiple
conformations of the ligand fitted into the density are shown in different
Third, we have presented the structure of hCE1 in complexes with an
anti-Alzheimer drug tacrine (Bencharit et al., 2003b).
The manner in which
tacrine binds, and the shape and size of hCE1 substrate binding gorge, are
distinct from the tacrine bound acetylcholinesterase structure determined
previously. Furthermore, tacrine appears in multiple
hCE1's substrate binding gorge. We conclude that hCE1's ability to bind the
same ligand in up to five conformations may contribute to its promiscuity and
facilitate its ability to process variety of endogenous and exogenous compounds
Diffraction data for the rCE, hCE1-homatropine, and hCE1-tacrine structures
were collected at SSRL Beam Lines 7-1, 9-1, and 9-2. Diffraction data for the
hCE1-naloxone structure was collected at the Advanced Photon Source (APS);
SER-CAT Beam Line 22-ID.
Redinbo MR, Bencharit S, Potter PM. Human carboxylesterase 1: from drug
metabolism to drug discovery. Biochem. Soc. Trans. 2003;31:620-4.
Bencharit S, Morton CL, Howard-Williams EL, Danks MK, Potter PM, Redinbo MR.
Structural insights into CPT-11 activation by mammalian carboxylesterases. Nat.
Struct. Biol. 2002;9:337-42.
Bencharit S, Morton CL, Xue Y, Potter PM, Redinbo MR. Structural basis of
heroin and cocaine metabolism by a promiscuous human drug-processing enzyme.
Nat. Struct. Biol. 2003a;10:349-56.
Bencharit S, Morton CL, Hyatt JL, Kuhn P, Danks MK, Potter PM, Redinbo MR.
Crystal structure of human carboxylesterase 1 complexed with the Alzheimer's
drug tacrine. From binding promiscuity to selective inhibition. Chem. Biol.