1. Human Esterases:
Chemical and Biochemical Considerations
Luke Lightning, PhD
1

2. Outline: Esterases and Carboxylesterases
• Introduction
– Esters
– Different esterases involved in drug metabolism
– Mechanism
– Biochemical Characteristics
• Human Carboxylesterases
– Molecular Structure 
• Overall Function
• Localization
• Substrate Specificity
• Evolutionary Relationships
2

3. Introduction: Esterases
• PubMed Search: at least 73 “different” human esterase genes
– complicated by duplicate entries
• / hydrolase-fold family (as of 9/28/04: 5440 sequences)
– Carboxylesterases (329 nucleotide sequences)
• Carboxylesterases (hCE-1, 2, 3) – broad substrate specificity
– Cholinesterases (114 nucleotide sequences)
• Acetylcholine esterase (AChE) – specific for acetylcholine (101
sequences)
• Butyrylcholine esterase (BChE) – broad substrate specificity (13
sequences)
– Juvenile Hormone Esterase – specific for hormone (12 sequences)
– Esterase D
– Lipases
• Others:
– Paraoxonases (Arylesterases)
3

4. Esterases vs Lipases
• water soluble substrates • insoluble or heavily aggregated
- short chain fatty acid esters - longer chain fatty acids esters
• activity correlates with [substrate] • activity correlates with substrate area
• more non-polar residues at the surface
• lid opening
Both:
• found in all kingdoms
• display broad substrate specificity
• overlapping protein sequence motifs
4

5. Clinical Consequences
• Activation (prodrugs)
• Inactivation (ester drugs)
• Inhibition  increased potential for drug-drug interactions
• Exposure to environmental pollutants or drugs  induction
• Induction  enhanced hydrolysis
5

6. Famous Esters
O OH
O CH3
O
aspirin
O O
O
Esther Rolle
O
n
polyester
6

7. Other Substrates for Esterases
O
CH3
H P
N H3C O
O NO2
N O
O
CH3
H3C
paraoxon
lidocaine CH3
N
heroin
O
O O
CH3 H3C
O O
7

8. Esterase Activity
O O
R2
esterase
+
R1 O R1 O
OH R2OH
H2O
acid alcohol
acid alcohol
part part
also can metabolize thiols, amides, and carbamates
8

9. Ester Hydrolysis
4-methylumbelliferone
acetate
CH3 CH3
O O
+
H3C O O O H3C OH HO O O
acetate
4-methyl-7-
acid alcohol hydroxycoumarin
part part
hCE-1 2,000
catalytic efficiency (kcat/KM):
hCE-2 60,000
9

10. Prodrugs and Esterases
• Prodrugs containing esters, amides, lactones 
– Increased solubility
– Increased bioavailability
– Less toxicity
• Various esterases activate prodrugs in humans
– CPT-11 (irinotecan)
• Used in the treatment of colon cancer
• Approved by the FDA in 3 days (1996)
• Acid form (SN-38) is a topoisomerase I inhibitor
• High interpatient variability in SN-38 pharmacokinetics
• some patients respond very poorly
• Tumor tissue from colon has lower level of esterase activity
• only 2% of SN-38 makes it to the tumor
• Gene therapy  enhance local production of SN-38 in tumors
10

11. CPT-11 (Irinotecan)
inactive
active
Km = 5 M
SN-38 CPT-11
topoisomerase I poison
no glucuronidation
pathway in tumors
- can give orally
inactive,
can be
inactive
recycled
11

12. General Mechanism
oxyanion hole
ESTER
ACID
12

13. P450 Activity
H H
O
Active site: H2O
ROH FeIII RH H2O
heme ferric,
low-spin
RH
ROH
FeIII
FeIII
Requires:
ferric, high-spin
e-
NADPH
lipid
HO
R . H2O .
O2 - RH
FeIV FeII
O2 ferrous
: :
: :
H2O2 . .
O2 = O-O
P450 reductase O RH O RH O RH
Cytochrome b5 FeIII FeIV FeV O-O RH - O-O RH.
.+
compound I ferryl oxene
FeII FeIII
“iron oxo” 2 H+ ferrous dioxy ferric superoxide
“oxy-P450”
e- (rate determining step)
H2O
H+ HO-O RH
-O-O
RH
.
- O-O RH
FeIII FeIII FeII
ferric H+ ferric ferrous
hydroperoxide peroxy superoxide
13

14. Crystal Structures: hCE-1 + tacrine
catalytic gorge
+ tacrine
14

15. Lovastatin
- cholesterol-lowering drug
- lipid soluble prodrug
- considerable inter-patient variability in therapeutic effect
- long term adverse effects include liver damage and myopathy
HO O
HO
COOH
O
OH
O
O
hCEs
H3C O
H3C O
H
H
CH3 CH3
CH3 CH3
H3C
H3C
lactone (inactive) -hydroxy acid form (active)
HMG-CoA reductase inhibitor
15

16. Lovastatin
• Approximate % of hydroxy acid formed by esterases in:
– Plasma 18%
– Liver microsomes 15%
– Liver cytosol 67%
• Genetic variation in esterase activity is suggested:
– 3 of 17 livers showed little or no
capacity for lovastatin hydrolysis
– inter-individual variation in lovastatin
hydrolysis by plasma esterase
16

17. Interspecies Variability: Esterase Activity
carboxylesterase activity in liver microsomes
17

18. Interindividual Variability
Protein Levels - Carboxylesterases
anti-rat carboxylesterase antibody used to determine protein content
10-fold variation
18

19. Genetic Polymorphisms: Esterases
48 Japanese individuals were screened for single-nucleotide polymorphisms in 9
esterase genes - J. Hum. Genet. 48, 249 (2003)
SNPs Polymorphisms
Arylacetamide deacetylase 23 1
Cholesterol esterase 117 15
Carboxylesterase 1 and 2 53 8
Esterase D 28 1
Granzyme A and B 22 1
Interleukin 17 11 0
Ubiquitin carboxyl terminal esterase 48 12
• 302 SNPs were identified in esterases  38 polymorphisms
• No variations in the catalytic triad
• Is there a correlation between genotype and phenotype?
• Do polymorphisms regulate induction?
• No analysis of BChE, paraoxonases, etc.
19

20. Esters Used to Test Human Variability
20

21. Interindividual Variability
esters amides thioester
21

22. Organophosphorus Pesticides
40 billion pounds of insecticides per year is used
CH3 O OH
S O
H3CO
O O esterase P
S O S O CH3
H3CO H3CO
acid
P
S O CH3
H3CO
malathion
CH3
P450
O O esterase
O O esterase
inactivation
H3CO
P
S O CH3
H3CO
malaoxon
22

23. Fatty Acyl Ethyl Ester Synthase/Esterase
esterase
(H2C)7 (CH2)7 (H2C)7 (CH2)7
OH OCH2CH3
ethanol
H3C H3C
cis-oleic acid
ethyloleate O
O
• fatty acid + ethanol  fatty acyl ethyl esters
– esterases play a role in cholesterol trafficking
– build-up in tissues of alcoholics  necrosis of organs
23

24. Transesterification
• cocaine + ethanol  cocaethylene (more lethal in mice)
• hCE-1: Km for cocaine = 116 M; Km for ethanol = 43 mM
• BChE: Km for cocaine = 12 M
• [cocaine] after 100 mg dose IV = 3 M
• [ethanol] in blood of people that have OD’d on cocaine = 7-110 mM
24

25. Sarin, Tabun, and VX gas: Biological Weapons
O CH3 O
P P CH3
F O CH3 H3C O N
H3C CH3
H 3C
O
CH3
Sarin N
P N CH3
Tabun H3C S
O
H3C
VX
CH3
AChE inhibitor – developed as a pesticide (1952)
most deadly nerve agent in existence
3X more deadly than sarin
300 g is fatal
"It's one of those things we wish we could disinvent."
- Stanley Goodspeed, on VX nerve agent
25

26. Serine Esterase Inactivation
Ser
Ser
OH O
O
O
P
H3C O
P
H3C O O
O NO2
O
paraoxon
H3C
H3C HO NO2
- hCE-1 is inactivated by these organophosphates
- point mutations in the active site of hCE-1
 efficient organophosphate hydrolase
- US government is developing variant forms of hCE-1 to treat personnel
at risk of exposure to biological weapons
26

27. Mammalian Carboxylesterases (CEs)
• located in the ER and cytosol of many tissues
• involved in detoxification or activation of:
– Drugs
– Environmental toxicants
– Carcinogens
– Fatty acid esters
• multiple isoforms exist in various animal species
• activate carcinogens  hepatocarcinogenesis
27

28. hCEs
• Human Carboxylesterases (hCEs)
– Originally classified on the basis of substrate specificity and pI
– However, they:
• are glycoproteins  different pI’s
• have overlapping substrate specificities
– Now classified based on sequence alignments:
• 3 groups for humans with 80% sequence identity within a group
• hCE-1 – “liver hCE”
• hCE-2 – “intestinal hCE”
• hCE-3 – “brain hCE”
– large interindividual variation (66-150X) in colon tumors
28

29. hCEs
• serine hydrolases
• can metabolize:
– esters, thioesters, amide-ester linkages
– carbamates
• localized in the ER and cytosol of many tissues
• glycosylation is essential for maximal catalytic activity
– probably assists in folding, solubility, circulatory t1/2
– unknown: if there is a tissue dependence on amount of hydrolytic activity
• hCE-1 activity in liver >> hCE-1 activity in heart
• importance in industry:
– prodrugs  active compound by hCEs
– major determinants of pharmacokinetic behavior
29

30. hCE substrates
• Xenobiotics:
– Anesthetics: cocaine, lidocaine
– Narcotics: heroin, meperidine
– Cholesterol lowering: lovastatin
– Angiotensin-converting enzyme (ACE) inhibitors: delapril,
imidapril, temocapril
– Anti-cancer: CPT-11
• Endogenous compounds:
– Fatty acid esters:
• short, and long chain acyl-glycerols, long-chain acyl-carnitine,
long-chain acyl-CoA esters
30

31. hCE Structure
• Hydrophobic N-terminus
– targets the protein to the ER
• HXEL-COOH at C-terminus
– retains the protein on the luminal side of the ER
• 3 amino acid “catalytic triad” (very similar to serine proteases)
– Ser, His, and Asp or Glu
• 4 cysteines
– Disulfide bonds
• N-linked glycosylation sites
31

32. Subcellular Organization of Membrane Bound hCEs
Lumen
S-S
Glu hCEs
His
Ser
N-linked
glycosylation S-S
sites
Phospholipid bilayer Cytoplasm
32

33. Carboxylesterases catalytic
oxyanion triad
hole
33

34. Other names:
• hCE-1
– Acyl coenzyme A cholesterol acyltransferase
– Monocyte/macrophage serine esterase 1
– Monocyte/macrophage serine esterase
– Alpha naphthylacetate esterase
– Brain carboxylesterase (hBr1)
– Cholesteryl ester hydrolase
– Liver carboxylesterase
– Carboxylesterase, liver
– Alveolar esterase
– Serine esterase 1
– Acid esterase
– Egasyn
– HMSE1, HMSE, ACAT, ANAE, SES1, CEH, HU1
• hCE-2
– Intestinal carboxylesterase
– Liver carboxylesterase 2
– iCE, CE-2
34

35. hCE-1 and hCE-2
• hCE-1 • hCE-2
– 568 Amino Acids – 623 Amino Acids
– 62,596 Da – 68,903 Da
• sequence identity
• sequence identity
– AChE 30%
– rabbit CE-2 73%
– rabbit CE-1 80%
• can activate CPT-11
– hCE-2 48% • high-affinity, high velocity
• can activate CPT-11 enzyme w/respect to CPT-11
• does not activate CPT-11
• deficiency may play a role in:
– rheumatoid arthritis
– non-Hodgkins lymphoma
35

36. hCE1
• is also present in monocytes and macrophages
• biological roles:
– chemoprotection of proteins in tissues - drug and xenobiotic
metabolism
– cholesterol trafficking within cells and between tissues
• fatty acyl ethyl ester synthase activity
• acyl-coenzyme A:cholesterol acyl transferase (ACAT) activity  cholesterol esters
• one of 3 cellular binding targets of tamoxifen  cholesterol lowering effects (????)
– protein retention and release from the ER
• complexes with UGTs and C-reactive protein to retain them in the ER lumen
36

37. Crystal Structures: hCE-1
• Philip Potter’s group (St. Jude’s, Memphis, TN, April 2003):
– in complex:
• with naloxone methiodide (heroin analog)
• with homatropine (cocaine analog)
• with tacrine (human AChE inhibitor (Ki = 38 nM), Alzheimer’s)
• large substrate binding gorge with rigid and flexible pockets
• binding gorge is lined with hydrophobic residues
• catalytic triad = Ser-221, His-468, Glu-354
37

38. Crystal Structures: hCE-1
hCE-1:tacrine
hCE1:naloxone
hexamer
trimer
38

39. hCE-1 Crystal Structure
oligomer analysis by AFM
monomers hexamers
trimers
39

40. hCE Tissue Localization
liver >> heart > stomach
spleen = testis = kidney
also present in plasma
liver > colon > SI > heart
liver clearance: both
kidney clearance: hCE-1
SI and colon clearance: hCE-2
40

41. hCE Induction
• In rats:
– Phenobarbital
– Aroclor 1254
– Polycyclic aromatic hydrocarbons
– Aminopyrine
– Clofibrate
– Pregnenolone 16- -carbonitrile
– Di(2-ethylhexyl)phthalate
– Not 3-methycholanthrene
– Testosterone, but not estrogen  sex differences?
41

42. hCE-1 Substrates
hCE-1
O meperidine
H3C
CH3
N
O
H3C N
O CH3
O
O
O
hCE-1
cocaine
H3C
hCE-1
O O
CH3
N
N
H delapril
O
O OH
42

43. hCE-2 Substrates
O
H3C
CH3 N N hCE-2
N
O
O
O
O CH3
O
cocaine hCE-2
(and BChE) CPT-11 N
N
O
CH3
N
HO
6-acetylmorphine O
H3C
O O
hCE-2
HO O
H3C
O
43

44. hCE-1
substrates
hCE-2
substrates
IN GENERAL: hCE-1: does not hydrolyze cmpds with bulky alcohol groups
hCE-2: does not hydrolyze cmpds with bulky acid groups
44

45. hCE-1 Binding
Compounds with more
hydrophobic R groups
(larger log P)
bind more tightly
(smaller Ki)
45

46. hCE-1 Substrate
Specificity
swap R3 and R4
swap R1 and R2
enantiomer
extend length of R1
hydrolysis products
remove R1
atropine
46

47. Cocaine Metabolism (hCE-1 and P450)
transesterification, hCE-1
• longer t 1/2
hCE-1 hCE-1 • more toxic
• higher
MAJOR brain:plasma
ratio
P450 P450 P450
hCE-1 hCE-1
transesterification, hCE-1
47

48. hCE-1 Substrate Specificity
in vivo
t1/2 (min)
3
30-40
--
• hCE-2 was the 1st human enzyme reported to hydrolyze 6-AM
• Km’s ( 6.8 mM) are > than in vivo [heroin]
• < 270 M in abusers; 3 M in patients treated for pain
• 1st order kinetics in vivo
• cocaine and heroin are metabolized by same enzymes
• “speedballing”  enhanced drug levels
48

49. Uses of hCEs
• Regulating hCE activity to treat narcotic abuse or overdose
• Regulating hCE activity to treat soldiers affected by sarin or
other biological weapons
• Directed Evolution
–  regio- and enantio-selective reactions in organic synthesis
• improved activity in organic solvent, high temperatures, acidic pH
49

50. Web and Meeting Information
• ESTHER database
– http://bioweb.ensam.inra.fr/ESTHER/general?what=index
• International Paraoxonase Meeting (1st, 2004)
– http://sitemaker.umich.edu/pons-conference
• International Cholinesterase Meeting (8th, 2004)
– http://www-b.unipg.it/~cholinpg/
50

51. Conclusions
• Wide variety of esterases present in humans
– different substrate specificity, localization, catalytic mechanism
• Esterases can act as hydrolases and synthases
– gaining prominence in the field of drug metabolism
• Interspecies and inter-individual variability in esterase activity exists
– does this affect drug metabolism?
– more studies needed:
• genetic polymorphisms
• Induction
• hCEs play important roles in the metabolism of drugs and endogenous cmpds
• Crystal structures are now possible
51